EP1576966A1

EP1576966A1 - Method of preparing a vaccine and anti-tumor vaccines

Info

Publication number: EP1576966A1
Application number: EP03815518A
Authority: EP
Inventors: Ling Room 08-48 Tian; Yuquan Wei
Original assignee: Shenzhen Tsinghua Yuanxing Bio-Pharm Science & Technology Co Ltd
Current assignee: Shenzhen Tsinghua Yuanxing Bio-Pharm Science & Technology Co Ltd
Priority date: 2002-12-27
Filing date: 2003-12-26
Publication date: 2005-09-21
Also published as: JP2006516258A; CA2511927A1; EP1576966A4; AU2003298984A1; WO2004067029A1

Abstract

The present invention provides a method of preparing a vaccine. The method includes: (1) analyzing specific antigen of particular pathogen; (2) obtaining a polynucleotide encoding the specific antigen; (3) obtaining a polynucleotide sequence having a sufficient difference from the polynucleotide; (4) preparing a vaccine using the polynucleotide sequence. The present invention also provides antitumor vaccines, particularly antitumor EGFR molecular vaccines. The antitumor EGFR molecular vaccines are a variety of new biotechnological vaccines constructed by using EGFR molecule as antigens, including protein vaccines, gene vaccines, virus vaccines, gene-modified vaccines and stably transformed symbiotic bacterium. One of the biological function of the EGFR molecular vaccines has an antitumor effect on a variety of solid tumor cancer which over-express the EGFR molecule, e.g., lung cancer, breast cancer, ovary cancer, colon cancer, prostate cancer, stomach cancer, etc., including a protective antitumor immune effect, therapeutic antitumor effect and inhibiting cancer metastasis. The antitumor action mechanism of EGFR molecular vaccine is that the EGFR molecule may destroy an individual's immune tolerance to self-EGFR molecule as cross-reactive antigens, and therefore induce an immuno-cross-response to the self-EGFR molecule in the individual, including active immune reaction (cellular immune reaction and humoral immune reaction) and passive immune reaction (adoptive immune reaction). The antitumor EGFR molecular vaccines are either traditional preparation (aqueous, lyophilized powder) or nano preparation prepared by nano biotechnology. The EGFR molecular vaccine exhibits an enhanced antitumor effect through the synergistic action of other immunopotentiator.

Description

FIELD OF THE INVENTION

The present invention is in the bio-technology field, more particularly, it relates to a method of preparing vaccines and its prepared bio-vaccines, especially to a novel anti-tumor vaccine, including anti-EFGR molecular vaccine. Molecular vaccines are in the field of new biotechnological drug, and encompass many aspects of biotechnology, such as PCR, Molecular Cloning, Gene Expression, Modem vaccine technology, and biotechnological medicine and pharmacology.

BACKGROUND OF THE INVENTION

Vaccine is a material that is capable of stimulating the organism immune system to initiate an immune response against specific target substances such as virus, bacteria and the like. Classical concept of vaccine originates from immunity against infection which mainly use treated microbial pathogen (such as virus, bacteria etc.) or its derivatives to immunize the organism so as to produce humoral immune response to prevent infectious diseases. For example, inactivated vaccine is derived by inactivating the active component of the infectious pathogen through chemical reaction. Attenuated vaccine is produced by mutating live virus or bacteria so that it cannot reproduce itself in biological organism. These two classes of vaccines achieve immunization through the surface protein (antigen) reacting with B and T gonorrhea cells. When a human is infected by pathogen, B and T gonorrhea cells can rapidly make response and eradicate it before the pathogeny organism produces destruction. However, these two vaccines have potential danger because they can be polluted by infectious pathogen. For example, there is always a faction of children being infected infantile paralysis because of being vaccinated against infantile paralysis. Therefore, one of development direction is subunit vaccine, also known as gene engineering vaccine or DNA recombinant vaccine, which is produced by DNA recombination technique and only consists of surface protein and initiates an immune response. It can be seen in Gene Engineering Principle, Edited by Ma Jiangang, published by Xi'an Jiaotong University Press, page 240, Nov. 2001. In addition to the aforesaid pathogen itself, its ingredient having immunogenicity and gene expression product, "naked" DNA is also studied to be used as gene vaccine of vaccine. "Naked" DNA consists of an antigen coding gene originated from pathogen and the plasmid DNA as its carrier. After the gene vaccine is introduced into human through injection or particle bombardment, or the like method, the gene fragment can synthesize antigen protein, and to initiate immune response. At present, the Expression-Library Immunization technique is the most systemic and objective means of finding immune active gene. It can be seen in the Fourth Tide: Bioeconomic, edited by Feng Zhanqi and Yang Tongwei, published by Economic Management Press, pages 169 to 171, 2000.
The main function of a vaccine consists in preventing disease. Now vaccine is also used to treat disease. Therefore, for normal human and animals, the function of vaccine is to enhance the ability of disease prevention and resistance of organism so as to prevent from diseases. For human and animal having certain disease or disorder, the function of vaccine is to induce organism to produce reaction aiming at specific pathopoiesia factor in order to eliminate focus and treat disease or disorder. It can be considered that vaccine is a general term of substance that can prevent, or treat, or prevent and treat disease or disorder by affecting the immune system of organism.
The immortalized cell resulted from out of control of cell division adjust in animal is known as tumor cell. The tumor having metastasis ability is known as malignancy, and the malignancy of epithelial tissue is known as cancer. It can be seen in Cell Biology, edited by Zhai Zhonghe, etc., published by Higher Education Press, pages 423 to 427, 2000 (2001 reprint). There have been a great number of fundamental research and clinical application researches about the origination of tumor and the response to tumor of host. About origination, it is shown that a great number of environmental factors have carcinogenicity and mutagenicity to animals. In fact, some tumors relate to exposure to certain specific substances (for exapmle mesothelioma of asbestos and shipyard worker). Known virus can also induce animal tumors, for example, hepatitis B virus relates to liver cancer. In the many cases, host produces immune response so as to resist tumor, while in some other cases, it may be protective.
The tumor cell and normal cell can be distinguished depending on the differentia of quality and quantity of their antigen. The antigen of the tumor cell, especially the tumor specific antigen being peculiar to tumor cell, becomes the research emphasis of tumor prevention and treatment.
Cancer vaccines for tumor treatment are of particular importance. This type of therapeutic vaccine is different from traditional preventive vaccine, in that, it is mainly used to treat patients who already suffer from cancer. The purpose of using cancer vaccine is to stimulate the patient's specific immune response against the tumor so as to achieve effective rejection eventually. The research and development of cancer vaccine has become a focus of tumor immunity treatment intemationally in recent years, and it mainly includes tumor cell vaccine, gene-modified vaccine, polypeptide vaccine, gene/DNA vaccine etc.
Tumor cell vaccines are intact, dead cells produced by treating tumor cells of a patient or sick animal with physical or chemical methods, such that after treatment, those cells will possess therapeutic or auxiliary therapeutic effect. The methods used to treat the tumor cells include X-ray radiation, or treating with organic solvent, etc. After introducing this vaccine to the organism by injection, or other methods, it can stimulate or enhance the patient's immunity against its tumor. Gene-modified vaccine, polypeptide vaccine and gene/DNA vaccines are all vaccines having therapeutic or auxiliary therapeutic effect on tumor, and made by using the tumor antigen or its fragments, or polynucleotides coding for tumor antigen or its fragments, and carriers or cells containing these polynucleotides.
As described above, vaccine plays an important role in disease prevention and treatment. With the aggravation of environmental pollution, the occurring frequency of tumor, as a serious abnormal growth of animal somatic cell, enhances. Therefore, it is necessary to develop new vaccine, especially cancer vaccine. The present invention provides a new method for making vaccine, and a new cancer vaccine-EGFR molecular vaccine, which meets the above requirements.

SUMMARY OF THE INVENTION

For the first aspect, the present invention provides a preparation method for vaccine. The method comprises:
1) analyzing specific antigen of particular pathogen;
2) obtaining a polynucleotide sequence encoding the specific antigen;
3) obtaining a polynucleotide sequence having a sufficient difference from the polynucleotide sequence; and
4) preparing the vaccine using the polynucleotide sequence obtained in step 3).
For the second aspect, the present invention provides a preparation method for cancer vaccine. The method comprises:
1) analyzing specific antigen of tumor cell in an animal;
2) obtaining a polynucleotide sequence encoding the specific antigen;
3) obtaining a polynucleotide sequence having a sufficient difference from the polynucleotide sequence; and
4) preparing the vaccine using the polynucleotide sequence obtained in step 3).
For the third aspect, the present invention provides a preparation method for EGFR vaccine. The method comprises:
1) analyzing EGFR antigen of tumor cell in an animal;
2) obtaining a polynucleotide sequence encoding the specific antigen;
3) obtaining a polynucleotide sequence having a sufficient difference from the polynucleotide sequence; and
4) preparing the vaccine using the polynucleotide sequence obtained in step 3).
For the forth aspect, the present invention provides a nucleic acid vaccine, which comprises a polynucleotide sequence that has certain differences with the polynucleotide sequence that encodes one specific antigen in a pathogen, and its homology needs to reach certain value. The homology between the polynucleotide sequence contained in the vaccine and the polypeptide encoded by the polynucleotide sequence of the specific antigen is 30-95%.
For the fifth aspect, the present invention provides a nucleic acid vaccine, which comprises two or more polynucleotide sequences that have certain differences with the polynucleotide sequence thate encode two or more specific antigens in a pathogen, and their homologies need to reach certain values. The homologies between the polynucleotide sequences contained in the vaccine and the polypeptides encoded by the polynucleotide sequences of the corresponding specific antigens are 30-95%.
For the sixth aspect, the present invention provides a protein or polypeptide vaccine, which comprises one protein or polypeptide molecule that has certain differences with the polynucleotide sequence of the protein portion of one specific antigen in a pathogen, and its homology is 30-95%.
For the seventh aspect, the present invention provides a protein or polypeptide vaccine, which comprises two or more protein or polypeptide molecules that have certain differences with the polynucleotide sequences of the protein portions of two or more specific antigens in a pathogen, and their homologies are 30-95%.
For the eighth aspect, the present invention provides an epidermal growth factor receptor (EGFR) nucleic acid vaccine, which comprises one polynucleotide sequence that has certain differences with the polynucleotide sequence that encodes an EGFR molecule gene in an organism, and its homology needs to reach certain value. The homology between the epidermal growth factor receptor encoded by polynucleotide sequence contained in the vaccine and the amino acid sequence in the epidermal growth factor receptor of the organism is 30-95%.
For the ninth aspect, the present invention provides an epidermal growth factor receptor (EGFR) nucleic acid vaccine, which comprises two or more polynucleotide sequences that have certain differences with the polynucleotide sequence that encode an EGFR molecule gene in an organism, and their homologies need to reach certain values. The homologies between the epidermal growth factor receptors encoded by the polynucleotide sequences contained in the vaccine and the amino acid sequence in the epidermal growth factor receptor of the organism are 30-95%. The two or more polynucleotide sequences contained in the vaccine may have different sources, and they may have different homologies with the polynucleotide sequence in the EGFR molecule gene of the organism.
For the tenth aspect, the present invention provides an epidermal growth factor receptor (EGFR) protein or polypeptide vaccine, which comprises one variant of EGFR molecule in an organism. The homology between the amino acid sequence of epidermal growth factor receptor contained in the vaccine and the amino acid sequence in the epidermal growth factor receptor of the organism is 30-95%.
For the tenth aspect, the present invention provides an epidermal growth factor receptor (EGFR) protein or polypeptide vaccine, which comprises two or more variants that are different with the EGFR molecule in an organism. The homology between the amino acid sequence of epidermal growth factor receptor contained in the vaccine and the amino acid sequence in the epidermal growth factor receptor of the organism is 30-95%.
For the eleventh aspect, the present invention provides a use of the epidermal growth factor receptor nucleic acid vaccine and the epidermal growth factor receptor protein or polypeptide vaccine in preventing and treating cancer.

DETAIL DESCRIPTION OF THE INVENTION

As used herein, "pathogen" refers to a foreign organism that invades an organism, including bacteria, virus, fungi, protozoon, helminth etc, which can invade other organisms and result in certain diseases or physiological conditions.
As used herein, "cancer cell" refers to a cell that proliferates and divides incontrollably, disengaging from the normal path of fade and death. The "cancer cell" population consists of a cancer organization. In the present invention, the term "cancer" is generalized, including any types of abnormal proliferation of a cell, and also involving cancer organization or cancer cell. It is required to inhibit the proliferation, or inhibit or remove these cells and organizations.
As used herein, "organism" refers to an animal, especially to "human". The method and vaccine of the present invention can be used in an animal with immune system.
The basic research results in the art or well-known methods can be used to confirm the specific antigen in the pathogen or cancer cell as the target of the vaccine of the invention. Since the abnormal cells in the specific pathogen or in vivo have specific label molecules, such as their specific antigen, the vaccine can be designed by taking these molecules as the target, to induce an immune response in the organism to the harmful substances, organisms or cells, so as to achieve a object for preventing and treating diseases.
According to public databases, such as GenBank, or many websites, the gene sequences or amino acid sequences of corresponding useful antigens can be found. According to central dogma, the sequences deduced from the amino acid sequences or gene sequences can also be used in the present invention. Useful websites includes, but not limited to,
http://www.sanger.ac.uk/Projects/Microbes.
http://bioweb.pasteur.fr/Genoist/TubercuList.
In the present invention, the nucleic acid sequences or amino acid sequences of polypeptide that are against the target antigen are different from the nucleic acid sequences or amino acid sequences of the protein or polypeptide portions that encode the target antigen. Their homologies are 30-95% of the coding products.
The homology is generally determined by sequence analysis software. The term "homology' in the context for two polypeptide sequences refers to two or more sequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same when compared and aligned for maximum correspondence over a comparison window or designated region. This is just as measured using any number of sequence comparison algorithms or by manual alignment and visual inspection.
For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
In the present invention, certain amino acid sequence in the antigen is chosen as the reference sequence, and the coding sequences of nucleotide sequences in the vaccine, or the amino acid sequences of proteins or polypeptides in the vaccine are testing sequences. The homology of the testing sequence and reference sequence is one aspect of the present invention.
If the nucleic acid sequence of target antigen or the amino acid sequence of polypeptide is taken as "parent sequence or reference sequence", the nucleic acid sequence or the amino acid sequence of polypeptide used in the present invention can be taken as "daughter sequence or derivative sequence".
The source of "parent sequence or reference sequence" can be the prior art, such as the databases mentioned above, websites, disclosed inventions and papers. They can also derived from the researches on specific antigen, either microbian antigen or cancer antigen in vivo. The parent sequence or reference sequence or its sequence information can be obtained by studying and analyzing the genome, chromosome, protein or polypeptide, mRNA, and DNA molecules.
The "daughter sequence or derivative sequence" is a sequence derived from the "parent sequence or reference sequence". It has some differences with the "parent sequence or reference sequence", but not completely different. The "daughter sequence or derivative sequence" can be existing sequence in nature, which has some structural correlation with the "parent sequence or reference sequence", or can be sequence obtained by artificial process. The former refers to the sequence that encoding homologic protein existed in different organisms or in individual organisms. For example, the coding sequence of human epidermal growth factor receptor, and the coding sequence of mice epidermal growth factor receptor, can be taken as the "parent sequence" and the "daughter sequence" as mentioned in the invention, respectively, or as the "reference sequence" and the "derivative sequence", respectively. These designates can be converted. For example, the coding sequence of mice epidermal growth factor receptor can be taken as the "parent sequence", while the coding sequence of human epidermal growth factor receptor can be taken as the "daughter sequence"; or the coding sequence of human epidermal growth factor receptor can be taken as the "parent sequence", and the coding sequence of mice epidermal growth factor receptor can be taken as the "daughter sequence". The "parent sequence or reference sequence" can be the initial substance, from which the "daughter sequence or derivative sequence" can be obtained by mutation or modification. The "daughter sequence or derivative sequence" that has some difference with the "parent sequence or reference sequence", can also be obtained by artificial synthesis processes. The nucleic acids, proteins and the fragmental substances thereof having "daughter sequence or derivative sequence", are obtained by searching corresponding databases, performing corresponding program on the computer to obtain the "daughter sequence or derivative sequence" of the "parent sequence or reference sequence", and then artificial synthesis. The "daughter sequence or derivative sequence" of the invention can also be obtained by screening corresponding molecule libraries. The nucleic acids, nucleic acid fragments, oligonucleotides, polynucleotides having "daughter sequence or derivative sequence", and the proteins, polypeptides, and fragments thereof having "daughter sequence or derivative sequence" can be used in the embodiments of the invention. In the embodiments of the invention, various modifications can be carried out on the nucleic acids, nucleic acid fragments, oligonucleotides, polynucleotides having "daughter sequence or derivative sequence", and the proteins, polypeptides, and fragments thereof having "daughter sequence or derivative sequence". It is also possible to include them in suitable vectors or cells or viruses, to achieve the object of the invention. The above-mentioned various substances used in the embodiments of the invention can be isolated, purified; or they can be mixed together, and even mixed with certain substances, such as adjuvant.
As used herein, the phrases "nucleic acid" or "nucleic acid sequence" refer to an oligonucleotide, nucleotide, polynucleotide, or to a fragment of any of these, to DNA or RNA of genomic or synthetic origin, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material, nature or synthetic origin.
The "coding sequence of' or "nucleotide sequence encoding" a particular polypeptide or protein", is a nucleic acid sequence which is transcribed and translated into a polypeptide or protein when placed under the control of appropriate regulatory sequences.
The term "gene" means the segment of DNA involved in producing a polypeptide chain. It includes regions preceding and following the coding region (leader and trailer) as well as, where applicable, intervening sequences (introns) between individual coding segments (exons).
"Amino acid" or "amino acid sequence" as used herein refer to an oligopeptide, peptide, polypeptide, or protein sequence, or to a fragment, portion, or subunit of any of these, and to naturally occurring or synthetic molecules.
The term "polypeptide" as used herein, refers to amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres, and may contain modified amino acids other than the 20 gene-encoded amino acids. The polypeptides may be modified by either natural processing, such as posttranslational processing, or by chemical modification techniques which are well known in the art. Modification can occur anywhere in the polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also a given polypeptide may have many types of modifications. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of a phosphytidylinositol, cross-linking cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristolyation, oxidation, pergylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, and transfer-RNA mediated addition of amino acids to protein such as arginylation. (See Creighton, T. E., Proteins - Structure and Molecular Properties, 2nd Ed., W. H. Freeman and Company, New York (1993); Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed., Academic Press, New York, pp. 1-12 (1983)).
As used herein, the term "isolated" means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally-occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.
As used herein, the term "purified" does not require absolute purity; rather, it is intended as a relative definition.
As used herein, the term "recombinant" means that the nucleic acid is adjacent to a "backbone" nucleic acid to which it is not adjacent in its natural environment. Backbone molecules according to the invention include nucleic acids such as expression vectors, self-replicating nucleic acids, viruses, integrating nucleic acids, and other vectors or nucleic acids used to maintain or manipulate a nucleic acid insert of interest.
"Recombinant" polypeptides or proteins refer to polypeptides or proteins produced by recombinant DNA techniques; i.e., produced from cells transformed by an exogenous DNA construct encoding the desired polypeptide or protein. "Synthetic" polypeptides or protein are those prepared by chemical synthesis. Solid-phase chemical peptide synthesis methods can also by used to synthesize the polypeptide or fragments of the invention. Such methods have been known in the art since the early 1960's (Merrifield, R. B., J. Am. Chem. Soc., 85: 2149-2154, 1963) (See also Stewart, J. W. and Young, J. D., Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Co., Rockford, I11, pp. 11-12) and have recently been employed in commercially available laboratory peptide design and synthesis kits (Cambridge Research Biochemicals). Such commercially available laboratory kits have generally utilized the teachings of H. M. Geysen et al, Proc. Natl. Acad. Sci., USA, 81:3998 (1984) and provide for synthesizing peptides upon the tips of a multitude of "rods" or "pins" all of which are connected to a single plate. When such a system is utilized, a plate of rods or pins is inverted and inserted into a second plate of corresponding wells or reservoirs, which contain solutions for attaching or anchoring an appropriate amino acid to the pin's or rod's tips. By repeating such a process step, i.e., inverting and inserting the rod's and pin's tips into appropriate solutions, amino acids are built into desired peptides. In addition, a number of available FMOC peptide synthesis systems are available. For example, assembly of a polypeptide or fragment can be carried out on a solid support using an Applied Biosystems, Inc. Model 431A automated peptide synthesizer. Such equipment provides ready access to the peptides of the invention, either by direct synthesis or by synthesis of a series of fragments that can be coupled using other known techniques.
A promoter sequence is "operably linked to" a coding sequence when RNA polymerase which initiates transcription at the promoter will transcribe the coding sequence into mRNA.
"Plasmids" are designated by a lower case "p" preceded and/or follow by capital letters and/or numbers. The starting plasmids herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids in accord with published procedures. In addition, equivalent plasmids to those described herein are known in the art and will be apparent to the ordinarily skilled artisan. In the present invention, some typical plasmids are constructed, as shown in Figure 2. These plasmids are one form of embodiments of the invention.
"Oligonucleotide" refers to either a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands which may be chemically synthesized. Such synthetic oligonucleotides have no 5' phosphate and thus will not ligate to another oligonucleotide without adding a phosphate with an ATP in the presence of a kinase. A synthetic oligonucleotide will ligate to a fragment that has not been dephosphorylated.
"Fragments" as used herein are a portion of a naturally occurring protein which can exist in at least two different conformations. Fragments can have the same or substantially the same amino acid sequence as the naturally occurring protein. "Substantially the same" means that an amino acid sequence is largely, but not entirely the same.
The term "variant" refers to "parent sequence or reference sequence" of the invention modified at one or more base pairs, codons, introns, exons or amino acid residues (respectively). Variants can be produced by any number of means including methods, for example, error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis, gene reassembly, GSSM, and any combination thereof.
For another aspect of the invention, said "daughter sequences or derivative sequences" can be taken as the "variants" of "parent sequences or reference sequences". Because they have some structural similarities with "parent sequences or reference sequences" in antigen, but also have some differences. Therefore, a material containing "daughter sequence or derivative sequence" may induce an immune response of the organism to the antigen, in particularly, may destroy the tolerance of the organism to the antigen that have already produced. Actually, it is indeed because that the material used in said vaccine of the invention has some similarity with the structure of the antigen, the immune response against the material in the vaccine of the invention, which is performed by the organism, may cross-react on the antigen. At the same time, since the material used in the vaccine of the invention has some differences with the structure of the antigen, the vaccine of the present invention is particularly useful in inducing a response of the organism against the antigen that has produced tolerance.
The method of the present invention is described by using EGFR molecular vaccine as an example. Actually, the present invention is not limited to EGFR molecular vaccine, and according to the present invention, a skilled person in the art can conveniently prepare other vaccines by using the method of the invention. The more important value of the invention is that it discloses a method for producing vaccines, especially a method that destroys the tolerance of an organism; especially it has important value in treatment of cancer.
The EGFR molecular vaccines are a variety of new biotechnological vaccines constructed by using homologous EGFR molecules derived from human or other heterogeneous biology that were reconstructed and evolved through modem biotechnological engineering or formed in biological evolution process as antigen, including recombined protein vaccine, recombined gene vaccine, recombined virus vaccine, genetically modified vaccine, and stably inverted symbiotic bacteria. These are cancer vaccines with novel concept.
A homologous EGFR molecule refers to the total designation of EGDR molecules from other species origins (such as, mice, rat, chicken, mackerel, drosphia, etc., see SEQ ID NO 1-14), which have some extent of similarities with human EGFR molecules at amino acid or nucleic acid level.
Human epidermal growth factor receptor (EGFR) is a trans-membrane, single chain, glycosylated protein with a molecular weight of 170KD. It consists of 1186 amino acid residues, and possesses Tyrosine Protein Kinase (PTK) activity. EGFR consists of 5 portions, including extracellular ligand-binding domain, transmembrane hydrophobic domain, juxtamembrane domain, PTK active site and receptor C-terminal peptide. EGFRs have various types of transcript means (See SEQ IN NO 1-5), and have various mutants, wherein the major mutant is type III EGFRvIII (also called ΔEGFR or de2-7EGFR, see SEQ ID NO 6), which is specific on the membrane of malignant tumor cell with highly tumor specificity. Human EGFR molecules are a type of gene family; other family members except for EGFR (also called c-erbB1/HER1) include c-erbB2/HER2, c-erbB3/HER3 and c-erbB4/HER4, which encoding membrane proteins p185erbB2, p160erbB3, p180erbB4 respectively. Although these members have highly homologous amino acid sequences and similar structural characteristics, their ligands are different. The target molecules of the cancer vaccine involved in the invention are EGFR (c-erbB1/HER1), excluding c-erbB2/HER2, c-erbB3/HER3 and c-erbB4/HER4.
The ligands of EGFR molecules, EGF or TGF, can act on EFGR through either autocrine pathway, or paracrine pathway, by activating PTK, and through a series of signal transduction, causing cells to divide and proliferate.
EFGR can be widely found on the surface of normal mammalian epithelial cell surface. On average, there are about 50-100 thousand receptor molecules per cell. Many cancer cells, such as lung cancer, breast cancer, ovary cancer, colon cancer, prostate cancer, bladder cancer, head and neck squamocarcinoma and glioma, all have over-expressed EGFR, up to 1-3×10⁶ molecules/cell. Take, as an example, in the case of lung cancer, the overexpression of EGFR is closely related to the cancer's infiltration, metastasis and prognosis. Logically, EGFR has been hailed as one of the ideal target molecules in cancer therapy. So far, however, the major application of using EGFR as a therapy target has been with monoclonal antibody and small molecular synthetic compounds. There has been some positive development in areas, such as using EGFRvIII molecule in tumor peptide vaccine, and anti-sense RNA gene therapy. Nonetheless, there have been few reports in literature regarding using EGFR as a target molecule in making anti-tumor molecular vaccine.
A tumor cell will produce one or more tumor antigens in its malignant convertion, proliferation process. However, in the most circumstances, the immunogenicity of the tumor cell is weak, and cannot induce an active immune response of the organism. From the viewpoint of immunology, a tumor cell is a "self' tissue cell in a host that can continuously express "normal" antigen (gene overexpression) and/or "abnormal" antigen (gene modification, mutation or deletion). Thus, the tumor antigen can be taken as a self-antigen. Under the normal conditions, the organism does not produce immune response to self-antigen, i.e., it presents an immune tolerance. In fact, self-antigen is the most relevant and abundant antigen, which needs to be tolerated by immune system of the host. The induction and maintenance tolerated by self-antigen is directed by various mechanisms, which can prevent the improper destruction to normal tissue. However, when organism sequestered antigen release occurs, or the change of self-antigen occurs due to biological, physical or chemical factors, it results in a self immune response, which leads to pathological damage and functional disturbance of the cells, tissues or organs where target antigens are located. Thus, if tumor cell sequestered self-antigen is released, or it performs certain changes, it is possible to induce a specific self immune response of the organism to the tumor cell; thus, the tumor is reduced or even extinct. As describe above, EGFR molecule overexpresses in various tumors, therefore, EGFR molecule can be taken as a tumor antigen and a self-antigen. Accordingly, various biotechnical modifications can be carried out on human EGFR antigen molecules (see SEQ ID NO 1-6), producing molecular vaccine, to induce anti-tumor immune response, and achieve an object of antitumorigenesis.
In a biological evolution process, there are large quantities of different extents of homological molecules between different species. For example, EGFR molecules are broadly present in biology, such as human, mouse, fruit flies, fishes, avians, and there are different extents of homologies between them. The homologies of human EGFR molecules and mice, chicken and drosphia EGFR molecules on amino acid level, are 90%, 64% and 32%, respectively. The slight difference formed by xenogeneic homological molecules in evolution process can be used to destroy immune tolerance, increase immunogenicity, induce self immune response of tumor cell, so as to achieve an object of antitumorigenesis. This is most likely that although the neutral mutation of homological gene in evolution process does not lead to a loss or change of its function, it may effect or change its means of immune response. When a xenogeneic homologous molecule is introduced into the body of a bearing subject, and expresses a corresponding xenogeneic homologous protein, the subject will recognize it as a foreign antigen, and will produce a corresponding antibody or cytotoxic lymphocyte to remove it. At the same time, since the expressed xenogeneic homologous protein has some extents of differences with corresponding protein molecule in the body of subject, it will produce non-specific cross-reaction to induce self-immune response, and destroy the immune tolerance of the organism to this protein.
As described above, the EGFR of human has different homologies with mice, chickens and fruit flies. The human EGFR has homology of 90% with mouse EGFR, 64% with chicken EGFR, and 32% with fruit flies EGFR analyzed by BLAST at the amino acid level. We use the immune cross-reactions produced by xenogeneic homologous EGFR moleculars to illicit self-immune reaction to break the tolerance of self-tumor cells. The result antibody competitively inhibits the combination of EGFR and its ligand, EGF or TGF- can inhibit the growth of tumor cells. In addition, the result can be obtained by the induced CTL.
Recent development in modem biotechnology provides a variety of techniques, such as error-prone PCR technology, DNA Shuffling, Phage Display technique, etc. to support EFGR molecular vaccine made by gene-directed evolution technique.
The invention relates to an anti-tumor EGFR molecular vaccine, including autologous EGFR vaccine, xenogeneic EGFR vaccine and directed evolution EGFR vaccine.
The invention relates mainly to a novel tumor vaccine---EGFR molecular vaccine with the xenogeneic homologous EGFR molecule origined from human or other animals as antigen, which is made either with modem genetic engineering, or derived from the natural evolutionary process. The vaccines of the present invention include protein vaccine, gene vaccine, virus vaccine, modified-gene vaccine and stable commensal bacteria. The EGFR molecular vaccine has effect to all the solid tumors wherein EGFR is over-expressed. The mechanism of anti-tumor effects is that EGFR molecule is an immune cross-reactive antigen, which can break the body's tolerance for self-EGFR molecule, and induce self cross-reactive immune response against the EGFR molecule. The anti-tumor EGFR molecular vaccine is also the nanoparticle agent which is made by the nanotechnology.

1. Origin of EGFR molecule, its selection and improvement

The term EGFR molecule as used herein includes autologous EGFR molecule, xenogeneic EGFR molecule, and gene-directed evolution EGFR molecule.
A key feature of the present invention is demonstrated in the use of xenogeneic homologous EGFR molecule as molecular antigen in anti-tumor immunotherapy. The EGFR molecular vaccine is derived through a natural evolutionary process, or made with modern genetic engineering.
EGFR is widely found in nature. It expresses in a variety of organisms ranging from mammals, such as human, mouse, to avian organism such birds and chickens, to lower organism such as nematode, and drosphia. The EGFR molecules from these different organisms have certain differences among them, and the homology among each other is from 30% to 100%. The homology between the xenogeneic EGFR molecules as disclosed in the present invention is from 30% to 95%. The exemplary species referred to are human, mice, chicken, and drosophila, and at the amino acid level, the homology of EGFR molecular of human with mice, chiken, and drosophila is, respectively, 90%, 64%, AND 32%. The human EGFR, mouse EGFR, chicken EGFR and drosophila EGFR molecules represent, respectively, the monitor points of homology related to the invention.
The EGFR molecule is immuno-tolerant by the host body, and thus its immunogenicity is very weak, if at all. By using a modem biotechnological engineering, autologous EGFR molecules can be modified with the procedure of gene directed evolution, and thus improved, thereby enhancing the immunogenicity of the EGFR antigen. The general technical protocol is using an error-prone PCR, random primer extension technique, and DNA shuffling technique to cause artificial mutation in autologous EGFR molecule and establish a gene mutation library. And then, a process of selection is conducted by using phage display technique, and ribosome display technique, to obtain EGFR molecules with stronger immunogenicity.
In addition, we can utilize the differences between the expression modification systems of bacteria, virus and other organisms, or further modify and improve the autologous EGFR molecules at the protein level to increase the immunogenicity of autologous EGFR.

2. EGFR Recombinant Gene Vaccine

Gene vaccine, also known as DNA vaccine, is a new type of vaccine based on nucleic acids made with modem molecular technology.
One of the EGFR molecular vaccines of the present invention is DNA vaccine. We search for sequences of EGFR disclosed in publicly available databanks, such as GenBank (including gene sequences, cDNA sequences, mRNA sequences and amino acid sequences). Using these sequences, we designed primers or probes, and using techniques such PCR, RT-PCR, molecular hybridization. From various commercial gene library, cDNA library, cell lines, tissue cultures, we cloned and isolated the intracellular domains of the EGFR molecules from a variety of different species. We have found that the extracellular domain of EGFR is the preferred active region causing immunogenicity. We can further use the gene directed evolution technique to select EGFR molecules with stronger immunogenicity. After confirming the sequences of the extracellular domain of the EGFR cDNAs using sequencing, we constructed Eukaryotic plasmid expression systems containing these intracellular domains of the EGFR using molecular cloning technique. These constructs are then transfected to CHO cell lines, and they are observed and tested for its EGFR expression and the level of expression. The recombinant EGFR constructs of these eukaryotic plasmid expression systems can be analyzed and confirmed using restriction enzyme digestion, SDS-PAGE and Western Blot. We used alkaline extraction to obtain confirmed recombinant EGFR expression plasmids, and then used ultracentrifuge, ultra-filtration methods to eliminate E.Coli endotoxins. After that, we got pure recombinant plasmid DNA. The plasmid DNA molecules can be used as DNA vaccine for immunization.
Fig. 2 shows some representative plasmids used to make the EGFR DNA molecular vaccine. The detailed process for constructing these plasmids is described briefly as follows: public database, such as GenBank, is used to obtain the cDNA sequences of EGFR molecules of human, mouse, chicken (which correspond respectively to SEQ ID NO 1-5, 7-9, 19). We designed the following primers based upon the cDNA sequences:
The total RNA from human lung cancer cell line A431, mouse lung cancer cell line LL2, and chick embryo are usedm, resoectively, to undertake RT-PCR amplification, and then collect and purify the amplified EGFR fragment (average 1.9 kb in length) using electrophoresis, and then subclone the PCR products. After confirming the sequences of the PCR subclones through sequencing, we digested them using Ncol and EcoRl, collected the 1.9 kb fragments and purifed them, and then inserted the fragments into the pORF-MCS vector (from the InvivoGen Corporation) which had been digested with Ncol and EcoRI, selecting recombiant plasmids. Candidate recombinant plasmids are confirmed by both restriction enzyme digestion analysis and PCR amplification. They are named pORF-hEGFR, pORF-mEGFR and pORF-chEGFR. For the construction of pcDNA-hEGFR, pcDNA-mEGFR and pcDNA-chEGFR, the process is similar to the above described process. The pcDNA3.1 (+) vector is made by InvitroGen, but the sequences of the PCR primers are varied somewhat from those primers as described in the above paragraph: They are, respectively:
Human EGFR gene's extracellular domain was used to construct and prepare the DNA vaccine for immunization of Lewis lung cancer model mouse. It was discovered that 8 weeks after immunization the survival rate of the mice injected with this EGFR DNA vaccine was 78%, significantly higher those mice injected with mouse EGFR DNA vaccine (with a survival rate of 25%), and much higher than comparision experienmental animals (with a survival rate of 10-15%). At the same time, we did not find any pathological changes to the lungs, liver, heart, and kidney of the experimental model mice. Further research was conducted and demonstrated that the induced self-autoimmune response in mice was mainly dependent upon CD4⁺ T lymphocytes. The CTL activity test did not find any target cells-specific cytocidal effect. Immunohistochemistry results showed deposition of autoantibodies in tumor tissues, but no such deposition in tumor tissues such as lung, liver, etc. The autoantibody in this case is mainly IgG.
The anti-sense RNA and RNAi molecules designed with EGFR molecule as template can be viewed as specific examples of the EGFR recombinant gene vaccine. This was not primarily by increasing the immunogenicity of EGFR molecule to induce anti-EGFR antibody and specific CTL reaction to achieve the goal of blocking the EGFR signal pathway, thereby further inducing tumor cell apoptosis, and inhibiting tumor cells growth and metastasis, but rather, it directly restricts and prohibits the expression of EGFR molecules at the DNA and RNA level.

3. EGFR Recombinant Protein Vaccine

Protein vaccine is a relatively traditional vaccine, but the protein possesses very good immunogenicity. One of the vaccines of the present invention is a recombinant protein vaccine, including those constructed with the recombinant proteins expressed by various expression systems, such as E.coli recombinant expression vectors, yeast recombinant expression vectors, baculovirus recombinant expression vectors.
Using molecular cloning technique, such as PCR, RT-PCR, molecular hybridization, we cloned and isolated the intracellular domains of the EGFR molecules of varieties of different species from various commercially available gene libraries, cDNA library, cell lines, and tissues, and further used the gene directed evolution technique to select EGFR molecules with strong immunogenicity. Then, the molecular cloning technique was used to construct prokaryotic plasmid expression systems, and transform suitable E.coli host with the result expression systems. Their EGFR expression levels were observed and examined. The recombinant EGFR constructs (plasmids) were analyzed and confirmed using restriction enzyme digestion, SDS-PAGE, and Western Blot. After confirmation, the recombinant EGFR molecules were used to transform E.coli, and the transformed E.coli cells were grown in a large quantity in order to produce recombinant proteins. Low temperature ultra centrifuge was used to collest the transformed cells, and then the cells were resuspended in PBS solution, and lysed using ultrasound techniques. The recombinant EGFR proteins were isolated and purified using ion-exchange chromatography and affinity chromatography. The isolated recombinant EGFR proteins can be used as protein vaccine for immunization.
Representative plasmids for recombinant EGFR protein vaccine expressed in E.coli are shown in Fig. 2B. The detailed process to construct these plasmids is described briefly as follows: the following PCR primers were designed in accordance with the cDNA sequences of EGFR molecules of human, mouse, and chicken described in the public databases, such GenBank, that correspond to SEQ ID NO 1-5,7-9,19, respectively.
Respective pORF-hEGFR, pORF-mEGFR and pORF-chEGFR were used as templates for PCR amplification. Then, the amplified EGFR fragments (average 1.9kb in length) were collected and purified using electrophoresis, and then the PCR products were subcloned. After confirming the sequences of the PCR subclones, they were digested using Ncol and BglII to collect the 1.9 kb fragments, and purified, and then the fragments were inserted into the pQE60 vector (from the Qiagen Corporation) which had been digested with two restriction enzymes Ncol and BglII, and the recombiant plasmids were selected. Candidate recombinant plasmids were confirmed by both restriction enzyme digestion analysis and PCR amplification. They are named as pQE-hEGFR, pQE-mEGFR and pQE-chEGFR.
Recombinant EGFR proteins can be produced by methods other than the E.coli recombinant expression systems as described above, such as, using the yeast recombinant expression system, baculovirus recombinant expression system.
See Figure 3 for the protocol of the construction process of the EGFR recombinant protein vaccine.
Respresentative maps for plasmid expressing EGFR recombinant protein vaccine made from yeast expression system are shown in Fig. 2C. The detailed procedure is described as follows: the following PCR primers were desiged in accordance with the cDNA sequences of EGFR molecules of human, mouse, and chicken stated in the public databases, such GenBank, corresponding to SEQ ID NO 1-5, 7-9, 19, respectively.
Respective pORF-hEGFR, pORF-mEGFR and pORF-chEGFR were used as templates for PCR amplification, and the amplified EGFR fragments (average 1.9kb in length) were collected and purifed using electrophoresis. And then the PCR products were subcloned. After confirming the sequences of the PCR subclones through sequencing, they were digested using two restriction enzymes Xhol and Xbal (in the case of plasmids containing mouse EGFR, Xhol and NotI double enzyme digestion were used), and the 1.9 kb fragments were collected and purified, and then inserted into pPICZ A vector (from Invitrogen Corporation) which had been digested with two restriction enzymes Xhol and Xbal (in the case of plasmids containing mouse EGFR, using Xhol and NotI double enzyme digestion). E.coli was transformed with these plasmid preparations, and the recombiant plasmids were selected. Candidate recombinant plasmids were confirmed by both restriction enzyme digestion analysis and PCR amplification. They are named as yeast expression plasmids: pYE-hEGFR, pYE-mEGFR and pYE-chEGFR. After digesting these yeast expression plasmids with Pmel to linerize them, an electric perforation method was used to transform yeast cell lines X33, or GS115. Zeocin resistance was used to select stable transformants. MMH (Minimal Methanol with histidine, MMH) and MDH (Minimal Dextrose with histidine, MDH) agar plate were used to determine and selecte Mut⁺ transformants. 6-10 Mut⁺ transformants were selected for small-scale expression, and then the methods of SDS-PAGE, Western Blot, ELISA, etc. were used to confirm the expressed recombinant protein. The Mut⁺ transformants with the highest expression efficiency were used as a large scale expression so as to establish yeast expressions seed libraries. Large flasks were used to culture or ferment the recombinant yeast cell lines, and the yeast pellets were collected using low temperature centrifuge. After the pellets were resuspended in PBS solution, ultrasound was used to break the cells. Then, ion exchange chromatography and affinity chromatography were used to isolate and purify the recombinant EGFR protein. The recombinant EGFR protein derived as such can be used as protein vaccine for immunization of subjects.
Similarly, yeast recombinant expression plasmids made with EGFR can be produced with other yeast expression systems.
Recombinant protein vaccine has a stronger effect than DNA vaccine in terms of the ability to illicit immune cross-reaction. It can stimulate production of high-titer anti-EGFR antibody and specific CTL activity to inhibit the growth and metastasis of tumor cells.

4. EGFR Recombinant Viral Vaccine

Recombinant viruses are also a good choice as a system for producing molecular vaccine. Molecular vaccines made with these recombinant viral expression systems include recombinant adenovirus vaccine, Lentivirus vaccine, adenovirus-related viral vaccine, retroviral viral vaccine, vaccinia virus vaccine and herpes simplex virus vaccine.
Currently, adenovirus vector is one of the most effective vectors in tumor gene therapy. It has the advantage of having high titer, safe, and can infect dividing, or non-dividing cells, and it does not integrate into the chromosomes of the host. In addition, adenovirus has relatively strong immunogenicity, which perhaps is a downside in gene therapy, but a big advantage in gene immunotherapy. As disclosed in the present invention, the recombinant adenovirus-derived EGFR recombinant viral vaccine is one of the most important embodiments. As described in the procedure above, we first cloned various kinds of autologous, xenogeneic or gene directed evolutionary EGFR cDNA, and then used these cDNAs to construct recombinant adenoviral expression vectors by molecular biological techniques. We then transfected 293 cells, and harvested the resulting recombinant adenovirus. The recombinant adenovirus was further confirmed using PCR, Western Blot, etc. We took the EGFR recombinant adenoviral vaccine made in large quantity using 293 cells, isolated and purified the recombinant virus using ultracentrifuge and ultra-filtration methods. The EGFR recombinant viral vaccine purified above can be used as vaccine to immunize subjects. See Figure 4A for the flow chart showing the construction of EGFR recombinant adenoviral vaccine.
We obtained the cDNA sequences of EGFR from human, mouse, and chicken stated in public databases, such GenBank, corresponding to SEQ ID NO 1-5,7-9,19, respectively. Based upon these sequences, we designed the following primers:
Respective pORF-hEGFR, pORF-mEGFR and pORF-chEGFR were used as templates for PCR amplification, and the amplified EGFR fragments (average 1.9kb in length) were collected and purified using electrophoresis. And then the PCR products were subcloned. After confirming the sequences of the PCR subclones through sequencing, we digested them using two restriction enzymes BglII and EcoRV double enzyme digestion, collected the 1.9 kb fragments and purified them, and then inserted the fragments into pShuttle-CMV vector (made by Quantum Biotechnologies) which was pre-digested with BglII and EcoRV. We selected recombiant plasmids. Resulting candidate recombinant plasmids were confirmed by both restriction enzyme digestion analysis and PCR amplification. They are named as Adenovirus shuttle expression plasmids: pShuttle-hEGFR, pShuttle-mEGFR and pShuttle-chEGFR. See Fig. 2D for plasmid maps of these Adenovirus shuttle expression plasmids. We then took the various adenovirus shuttle expression plasmids as described before, digested them with Pmel enzyme, and co-transformed E.coli BJ5183 cells with backbone vector containing the adenovirus genome pAdEasy-1 or pAdEast-2. The resulting recombinant adenovirus vector plasmids are named as pAd-hEGFR, pAd-mEGFR and pAd-chEGFR. These adenovirus vector plasmids were digested with Pacl enzyme, and then using the Calcium-phosphate-DNA coprecipitation method, they were transfected into the adenovirus packaging cell line 293 cells. The resulting recombinant Adenoviruses are called Ad-hEGFR, Ad-mEGFR and Ad-chEGFR. PCR, Western blot and other methods were used to confirm that the EGFR gene had been indeed integrated into the adenovirus vector and efficiently expressed in Eukaryotic cells.
Depending upon the difference of the adenovirus genome, the recombinant adenovirus vaccine can be classified into two groups: the first group is called the generation I of EGFR recombinant adenovirus, resulting from the recombination between adenovirus shuttle expression plasmid pShuttle-EGFR and AdEasy-1 recombinant, thus named Ad-hEGFR I, Ad-mEGFR I and Ad-chEGFR I, and the second group is called the generation II of EGFR recombinant adenovirus, resulting from the recombination between adenovirus shuttle expression plasmid pShuttle-EGFR and AdEasy-2 recombinant, thus named Ad-hEGFR II, Ad-mEGFR II and Ad-chEGFR II.
In addition, the EGFR recombinant virus vaccine can be modified specifically to increase its targeting ability with the compound Mannan.
Because adenovirus can introduce genes effectively, EGFR recombinant viral vaccine made with adenovirus can effectively induce the anti-tumor immune response in the subject, thereby inhibiting the growth of tumors with over-expressed EGFR.
Lentivirus vector is a new generation gene therapy vector. It is originated from lentivirus which has a HIV-1 replication deficiency. It is different from the traditional reverse transcriptase virus vector originated from the Moloney Leukemia Virus (MoMLV). The difference is that Lentivirus vector can effectively transfect both dividing and non-dividing mammalian cells, and it has good biological safety. In addition, Lentivirus vector differs from Adenovirus vector in that Lentivirus vector can integrate into the host cell's chromosomes, and thus enable the introduced exogenous gene's stable and long-term expression. Recombinant Lentivirus vector vaccine is also an important variation of EGFR recombinant virus vaccines. The process to make EGFR Recombinant Lentivirus Vaccine is similar to what is described with respect to adenovirus. That is, we first cloned various autologous, xenogeneic, or gene-directed evolution EGFR cDNA, and then using molecular biology technique, we constructed its recombinant Lentivirus expression vector, then transfected 293FT cells, resulting in recombinant Lentiviruses. The recombinant Lentiviruses were confirmed using PCR, Western Blot, etc. Further, after confirmation, we used 293FT cells to amplify large quantity of the EGFR Recombinant Lentivirus vaccine, and further isolated and purified the recombinant Lentivirus using ultracentrifuge and ultrafiltration. The purified EGFR Recombinant Lentivirus can be used as vaccine to immunized subjects. See Fig. 4B for a flow chart of the construction of Recombinant Lentivirus vaccine.
We used the ViraPower™ Lentiviral Gateway® Expression Kit made by Invitrogen Corporation to produce EGFR recombinant Lentivirus. The detailed process is as follows: we first searched public databases, such as GenBank, to obatin cDNA sequences of EGFR molecule of human, mouse and chicken. Their sequences are listed as SEQ ID NO 1-5, 7-9, 19, respectively. We then designed the following PCR primers:
We used pORF-hEGFR, pORF-mEGFR and pORF-chEGFR as templates for PCR amplification, then collected and purified the amplified EGFR fragments (average 1.9kb in length) using electrophoresis, and then subcloned the PCR products. After confirming the sequences of the PCR subclones through sequencing, we digested them using two restriction enzymes Ncol and EcoRV double enzyme digestion, collected the 1.9 kb fragments and purify them, and then inserted the fragments into pENTR11 vector (made by Invitrogen) which was pre-digested with Ncol and EcoRV. We selected recombiant plasmids. The resulting candidate recombinant plasmids were confirmed by both restriction enzyme digestion analysis and PCR amplification. They are named as pENTR-hEGFR, pENTR-mEGFR and pENTR-chEGFR. See Fig. 2E for plasmid maps of these Lentivirus expression plasmids. We then took the various Lentivirus expression plasmids as described before, transformed E.coli DH5 cells together with backbone vector containing the Lentivirus genome pLenti6/V5-DEST. The resulting recombinant Lentivirus vector plasmids are named as pLenti-hEGFR, pLenti-mEGFR and pLenti-chEGFR. See also Fig. 2E. We then mixed these Recombinant Lentivirus vector plasmids with packaging mixture (the ViraPower™ Packaging Mix), and then using the Calcium-phosphate-DNA coprecipitation method, the Recombinant Lentivirus vector plasmids were transfected into the Lentivirus packaging cell line 293FT cells. The resulting recombinant Lentivirus is called Lenti-hEGFR, Lenti-mEGFR and Lenti-chEGFR. PCR, Western blot and other methods were used to confirm that the EGFR gene had been indeed integrated into the Lentivirus vector, and that EGFR had been correctly, and efficiently expressed in Eukaryotic cells.

5. Modification and Improvement of EGFR Molecular Vaccine

What is described above is the basic procedure of preparing recombinant vaccine (including recombinant gene vaccine, recombinant protein vaccine and recombinant virus vaccine), and in the preferred embodiment, recombinant EGFR molecular vaccine. We now describe more embodiments hereinafter which include modifications and improvements that can enhance the efficency and specificity of the EGFR molecular vaccine.

1) Choice of adjuvant for the EGFR molecular vaccine

Suitable adjuvant is good carriers for vaccines that can increase the effective immune response of the vaccine. Different vaccines have different adjuvants. In the present invention, we used Freund's adjuvant and liposome for the EGFR DNA vaccine. For the EGFR recombinant protein vaccine, we mainly used aluminum adjuvant, and for the EGFR recombinant viral vaccine, generally, no adjuvant was used.

2) EGFR Gene-Modified Vaccine

Gene-modified vaccine refers to cell vaccine derived from human, other xenogeneic organism's cell lines transfected with EGFR molecule. These cell vaccines include vaccine made using various stably transformed tumor cell lines, vascular endothelial cells and dendritic cells. They also include vaccines made by using tumor cells or tissues infected by recombinant viruses. EGFR gene-modified vaccine is a particular example of EGFR molecular vaccine.
As discussed above in the present invention, EGFR is highly expressed in a variety of tumors, such as lung cancer, breast cancer, ovarian cancer, colorectal cancer, prostate cancer, stomach cancer, bladder cancer, head and neck squamocarcinoma, and glioma, etc. However, EGFR is tolerated in all these tumors, and thus cannot elicit immune response. Nonetheless, by using gene-modified vaccine, we can break the immune tolerance in these cells, and thus induce anti-EGFR immune response. We used Eukaryotic plasmids containing recombinant EGFR from a variety of different sources to transfect various kinds of tumor cell lines, such as lung cancer line A431, breast cancer cell line MCF7 etc., or vascular endothelial cells. After transfecting, we selected stable transfectants containing stably expressed EGFR molecules. We harvested the transfectants, and generated cellular vaccines using paraformaldehyde fixation method. In addition, we can also use various EGFR recombinant viral vaccines from different sources to infect tumor cell lines, tumor tissue, and tumor vascular endothelial cells, etc. We then can generate cellular vaccine using the same paraformaldehyde fixation method.

3) Stable EGFR transformants of commensal bacteria

EGFR molecular vaccines also include live vaccine, which is the stable EGFR transformants of commensal bacteria.
To produce these live vaccines, we used prokaryotic recombinant expression plasmids constructed with EGFR from various different sources, such as autologous, xenogeneic, and gene-directed evolutionary EGFR. We used these plasmids to transform intestinal commensal bacteria, such as Bifidobacterium. We then selected stable transformants. These stable EGFR transformants of commensal bacteria can continuously secrete exogenous EGFR molecules, thereby inducing the body's immune response. Thus, they are categorized as live vaccine. It is a particular example of the EGFR molecular vaccine.

4) Targeted Nano particles for EGFR molecular vaccine

The pure recombinant EGFR molecular vaccine (including EGFR recombinant gene vaccine, recombinant protein vaccine and recombinant viral vaccine) still should improve to enhance its specificity and immunogenicity. The present invention discloses the use of nanotechnology to enhance and modify the EGFR molecular vaccine, by using targeted nanoparticles in making the EGFR molecular vaccine. Using EGFR molecules, either its DNA or protein form, as target antigen molecule, biological molecules such as liposomes, degradable polymer biological materials, such as poly DL-lactide-co-glycolide polymer (PLGA) as nano materials, MIP-3 and Flt3-L as modifying genes, dendritic cells as target cells, we can construct a new type of targeted EGFR vaccine nanoparticles.
The EGFR molecular vaccine targeted with nanoparticles in the present invention come in a variety of forms, including: (1) mannan-modified nanoparticle, including mannan-modified recombinant adenoviral EGFR vaccine and protein vaccine, mannan-modified liposome recombinant EGFR gene vaccine and protein vaccine, (2) gene-targeted nanoparticle, including gene-targeted nanoliposome recombinant EGFR vaccine, gene-targeted nano-PLGA recombinant EGFR vaacine, and gene-targeted recombinant adenoviral EGFR vaccine, which express EGFR and MIP-3 simultaneously, (3) recombinant adenoviral EGFR vaccine targeting cancer vescular endothelial cell, including RGD-modified recombinant adenoviral EGFR vaccine).
As disclosed in the present invention, the diameter of the nanoparticles is generally less than 500nm, and they can be classified into three sizes: 200-500nm, 100-200nm, and 50-100nm. It is preferable to use nanoparticles with diameter ranging from 50nm to 100nm, and the nano peak value at around 80nm.
The following procedure applies to the preparation of Mannan-modified Recombinant Adenovirus EGFR vaccine: we first used the conventional method to obtain and amplify EGFR Recombinant Adenovirus (either Generation I or II), and then used chromatography or ultracentrifuge to purify the recombinant adenovirus. Next, we dissolved 70mg Mannan from Sigma into 5ml 0.1M phosphate buffer (pH6.0) to reach a final concentration of 14mg/ml, and then added 45ml 0.01 M Sodium Periodate solution, and mixed and oxidized it at 4□ for 60 minutes. After that, we added 10µl glycol, and incubated for 30 minutes at 4□, resulting in Oxidative Mannan (Ox-M) mixture. We then loaded the Ox-M mixture onto Sephadex-G25 columns previously balanced with bicarbonate buffer (pH6.0-9.0) and performed chromatography, with Ox-M being eluted into 2ml sized empty vessel. After that, we mixed the purified Ox-M with 1×10¹⁴ recombinant adenovirus particles at room temperature overnight, obtaining the needed Ox-M-Adenovirus. We then added 1mg/ml Sodium Borohydride to the Ox-M-Adenovirus, left it at the room temperature for 3 hours, forming Reductive Mannan Adenovirus (Red-M-Adenovirus). Both Ox-M-Adenovirus and Red-M-Adenovirus are desalted by ultrafiltration, and condensed, filtering out bacteria. They were then stored in small test tubes, and preserved at -800. The Mannan-modified Recombinant EGFR Adenovirus can be used as vaccine to immunize a subject.
As the size of the adenovirus is around 80nm, it is considered a natural nanoparticle. Adenovirus particles can be modified so that the adenoviral fiber protein can express the tri-peptide: RGD. The RGD tripeptide has specific ability to target tumor vascular endothelial cells. Recombinant Adenovirus EGFR vaccine modified with RGD can be viewed as a natural targeted EGFR molecular vaccine nanoparticle.
In the present invention, the AdEasy system is utilized to construct the RDG modified Adenovirus Recombinant EGFR vaccine. The detailed process is shown step by step in Figure 5. The detailed process is as follows: we digested the Adenovirus genome backbone plasmid pAdEasy-1 and pAdEasy-2 with restriction enzyme Spel (Sp), and then used T4 DNA Polymerase to fill in the ends (filling, f) so as to make them blunt, and then digested the filled-in product with PacI (P), and recovered the 6211bp and 3579bp fragments using electrophoresis, and named them as AdFiber I/Sp/f/P and AdFiber II/Sp/f/P, respectively. These fragments contain the intact Adenovirus fiber protein gene. Separately, the pSuttle vector was prepared by digesting it with BamHI first, and then filling in with T4 DNA Polymerase, and then digested with PacI. After such BamHI/filling /PacI-digestion treatment, the vector is ready to be inserted with the AdFiber I/Sp/f/P and AdFiber II/Sp/f/P fragments. The resulting recombinant plasmids are named pSh-AdFiber I and pSh-AdFiber II, respectively. We then digested pSh-AdFiber I with Nhel enzyme, filled in with T4 DNA polymerase, and digested them again with Kpnl enzyme (NheI/filling/KpnI), recovered, using electrophoresis, the 2090 bp fragment called AdFiber I/Nh/f/K; inserted this fragment into a pUC18 vector which had been pre-digested with Smal and Kpnl double enzyme digestion, resulting in the recombinant plasmid named pUC-AdFiber I. On the other hand, pSh-AdFiber II was digested with AvrII enzyme, then filled in with T4 DNA polyerase, and then digested with HindIII (AvrII/filling/HindIII), using electrophoresis, to recover the 838 bp fragment, called AdFiber I/A/f/H. This fragment was inserted into a pUC18 vector which had been previously digested with Smal and HindIII double enzyme digestion, resulting in new plasmids named pUC-AdFiber II. Then, we designed a series of PCR primers so as to use pUC-AdFiber I and pUC-AdFiber II as templates to amplify the Adenovirus knob, (Ad-knob) gene sequences. The primers used are, respectively:
Using primers F1-R1, F2-R2, F3-R1 and F2-R3, respectively, for the first round PCR, we obtained products PCR1, PCR2, PCR3 and PCR4. Again, using F1-R2 and F3-R3 as primers, and using the amplified products from the first round, PCR1 and PCR2, PCR3 and PCR4 as templates, we undertook the second round of PCR amplification, resulting in PCR products PCR1-PCR2 (PCR I), PCR3-PCR4 (PCR II). We took the PCR I and PCR II from the amplification in the second round, inserted them into pBR322 vector that had been previously cut with EcoRV. The resulting recombinant plasmids are named pBR-PCR I and pBR-PCR II. The sequence of the RGD-4C duplex is as follows:
We inserted the RGD-4C into the pBR-PCR I and pBR-PCR II vectors, where the vectors were previously digested with Smal. The resulting recombinant plasmids are named pBR-PCR/RGD I and pBR-PCR/RGD II. The sequences of the recombinant plasmids are confirmed using sequencing. Cut pBR-PCR/RGD I with NheI/KpnI double digestion , using electrophoresis, recovered the PCR/RGD I fragment, and inserted the fragment into the pUC-AdFiber I vector which had been double digested using NheI/KpnI. The resulting recombinant plasmids are called pUC-AdFiber-RGD I. Similarly, we used AvrII/HindIII to double digest pBR-PCR/RGD II, and using electrophoresis, recovered the PCR/RGD II fragment, and again inserted the fragment into pUC-AdFiber II vectors previously digested with double enzume AvrII/HindIII. The resulting plasmid is named pUC-AdFiber-RGD II. Afterwards, we used Spel/Pacl double enzyme to digest pUC-AdFiber-RGD I and pUC-AdFiber -RGD II vector, used electrophoresis to recover the AdFiber-RGD I and AdFiber-RGD II fragments, and then inserted the fragment into pAdEasy-1, pAdEasy-2 vectors both of which were previously digested with SpeI/PacI. The resulting recombinant plasmids are called pAdEasy-RGD I, and pAdEasy-RGD II, respectively. Then, we first took Adenovirus shuttle expression plasmids pShuttle-hEGFR, pShuttle-mEGFR and pShuttle-chEGFR as described above, and linerized them with Pmel. After that, we co-transformed the E.coli BJ5183 cells with these Shuttle-EGFR plasmids together with pAdEasy-RGD I, and pAdEasy-RGD II, respectively. The resulting recombinant plasmids are named Adenovirus plasmids pAd-RGD-EGFR I, and pAd-RGD-EGFR II. We used the Adenovirus plasmid pAd-RGD-EGFR I to transfect 293 cells, resulting in recombinant Adenovirus named Ad-RGD-EGFR I. Similarly, we used the Adenovirus plasmid pAd-RGD-EGFR II to transfect 293E4pIX cells, resulting in recombinant Adenovirus named Ad-RGD-EGFR II. After purification, Ad-RGD-EGFR I and Ad-RGD-EGFR II can be used as vaccine to immunize subjects, and more importantly, these vaccines possess specificity targeting tumor vascular endothelial cells.
It is found in the present invention that EGFR molecular vaccine targeted with nanoparticles can increase the immunogenicity of EGFR greatly compared to a normal EGFR molecular vaccine, and thus enable EGFR to illicit even stronger anti-tumor immune response. See Fig. 6 for schematic description of the mechanism of action for EGFR molecular vaccine targeted with nanoparticle.

5) Combination of EGFR molecular vaccine with other immune response stimulating factors

The anti-tumor effect of EGFR molecular vaccine can be enhanced by the combing effects of other immune response stimulating factors. These factors include cytokines, such as IL-2, TNF, IFN- , and GM-CSF, etc., chemokines, such as MIP3, MIP3, and IP10, etc., stringent factors, such as CEA, HSP70, HSP90, etc., and various immune co-stimulating factors, such as B7, etc. The combinatorial effects of these immune response stimulating factors with EGFR molecular vaccine can realize their effects through gene fusion at the gene level, or fusion protein at the protein level. They can also act through co-transfecting tumor cells, dendritic cells, and vascular endothelia cells at the cellular level.

6. The anti-tumor effect of EGFR Molecular Vaccine

As discussed above, the present invention provides a new type of molecular vaccine using tumor associated proteins. As in the specific embodiment of EGFR molecular vaccine, it encompasses protein vaccine, gene vaccine, viral vaccine, and gene-modified vaccine, all made using autologous, xenogeneic, or gene directed evolutionary EGFR molecules. These molecular vaccines possess effective anti-tumor functions, including preventing tumor formation, inhibiting tumor growth, anti-metastasis effect on tumor, and increasing the survival rate of human or animals carrying tumors.
To observe the anti-tumor effect of EGFR recombinant DNA vaccine, mice were randomly divided into groups (15 mice per group), and each group was injected intramuscularly 100µg EGFR recombinant DNA vaccine, hEe-p, mEe-p, c-p (control plasmid), or Saline, respectively. The injection was done once a week for four weeks continuously. One week after the fourth immunization, immunized mice were each implanted with 5×10⁵ of LL/2c Lewis lunger cancer cells (Fig. 7A and C), or MA782/5S mammary cancer cells (Fig. 7B and D), respectively. It can be seen from the figures that tumors kept growing in mice immunized with mEe-p, c-p, or Saline, while mice immunized with hEe-p exhibited significant protective immunity. In addition, the survival rate of hEe-p immunized mice is significantly higher than those of the mice immunized with mEe-p, c-p, or saline. For example, mice immunized with hEe-p lived longer than 5 months. After 150 days of tumor implantation, the mice immunized with LL/2c Lewis lung cancer and with MA782/5S mammary cancer reached survial rate of 60% and 66%, respectively. It is also discovered that the protective immunity is dosage-dependent. That is, the immunity obtained with dosage of 150µg is similar to that of 100µg of the EGFR DNA vaccine, but there is almost no immunity when the dosage is lowered to 5-15µg. In addition, the mice bearing EGFR-negative tumors, such as H22 liver cancer, and MMT-06052 mammary cancer exhibited no protective immunity when immunized with hEe-p.
Other than the protective immunity, EGFR recombinant DNA vaccine also possesses therapeutic immunity (Fig. 8). Likewise, mice were randomly divided into groups (15 mice per group). They were each injected subcutaneously once a week for 4 continuous weeks with 1×10⁶ LL/2c Lewis Lung cancer cells (Fig. 8A and C), or MA782/5S mammary cancer cells (Fig. 8B and D). Five days later, they were injected intramuscularly 100µg each hEe-p, mEe-p, c-p, or saline once a week for 4 continuous weeks. As seen from the figures, tumors in the mice immunized with mEe-p, c-p, or saline continued to grow, while tumors in the mice immunized with hEe-p exhibited significant therapeutic effect. In addition, the survival rate of the mice immunized with hEe-p is significantly higher than those immunized with mEe-p, c-p, or saline. The mice immunized with hEe-p lived longer than 5 months. 150 days after tumor implantation, the mice carrying LL/2c Lewis cancer and MA782/5S mammary cancer have a survival rate of 40% and 53%, respectively.
EGFR recombinant protein vaccine has a similar protective and therapeutic immunity. See Fig. 9. As described above, 6-8 weeks old female mice which carried C57BU6 or BALB/c were randomly divided into groups, and tumor models were established for LL/2c Lewis lung cancer, MA782/5S mammary cancer and C26 intestinal cancer in those mice. Tumor-bearing mice were injected subcutaneously EGFR recombinant protein vaccine in the amount of 5-50µg, or adjuvant aluminium hydroxyl gel and saline 100µl, once a week for 4 consecutive weeks. The mice immunized with recombinant chEGFR protein vaccine exhibited significant anti-tumor protective immunity and therapeutic immunity, and the growth of the LL/2c Lewis lung cancer and MA782/5S mammary cancer is inhibited, and the life span of the mice bearing these two tumors is extended, while the EGFR-native tumor C26 is not significantly affected. In addition, it can be seen that tumors kept growing at a fast pace in the control groups where the mice were immunized with recombinant mEGFR protein vaccine, alum adjuvant, or saline, and the life span of these mice were significantly shortened. Comparing to the control group, the tumor volume (t-test) and the length of survival (log-rank test) in the test groups showed significant difference (p<0.05). Fig. 9 shows the anti-tumor effect of EGFR recombinant protein vaccine in the mice bearing MA782/5S mammary cancer.
Metastasis is a common cause for tumor progression and for failure of chemothearpy and radiation therapy. The presence of tumor cells in blood and lymph circulation and the formation of microtransferer is a key to metastasis. It is disclosed in this invention that recombinant EGFR molecular vaccine has anti-metastasis effect on tumor. See Fig. 10. It is discovered during the therapy research of LL/2c Lewis lung cancer metastasis in animal model by injection in the tail vein, tumor-bearing mice immunized with chEGFR protein vaccine has far less metastasis in lung, or to a much lesser degree comparing to control group, whereas tumor-bearing mice in the control groups where they were immunized with recombinant mEGFR protein vaccine, alum adjuvant, or saline showed 100% metastasis, and they showed countless transferer. Figure 10 shows the anti-tumor metastasis effect of EGFR recombinant protein vaccine in mice bearing LL/2c Lewis lung cancer. From the figure, it can be seen that the number of transferer of LL/2c lung cancer and lung wet weight after LL/2c tumor metastasis in the mice immunized with chEGFR protein vaccine are both less comparing to control group.
In addition, EGFR molecular vaccine can inhibit growth of tumor cells in vitro, and they possess adoptive anti-tumor immunity in vivo. See Fig. 11. The process is described as follows: mice were immunized with EGFR DNA vaccine, and sera were collected 7 days after the fourth immunization. Serum Immunoglobulin (Ig) was purified using affinity chromatography. Various conentration of Ig (1-1000 mg/ml) were added to 2×10⁵/ml EGFR-positive tumor cells (A549, LL/2c, MA782/5S) and EGFR-negative tumor cells (H22 and MMT-06052), both kinds of tumor cells being in log-growth stage. The cells and the Ig were then co-cultured for 72 hours, and live cells were examined using Tai Pan Blue method, and the rate of growth inhibition was calculated. The results showed that EGFR-positive tumor cells that were treated with Ig purified from the sera of mice immunized with human EGFR recombinant DNA vaccine hEe-p showed significant growth inhibition, whereas EGFR-negative tumor cells were not affected. See Fig. 11A. As a control, Ig derived from non-immunized normal mice was co-cultured with the corresponding tumor cells. It has no effect on either EGFR-positive tumor cells, or EGFR-negative tumor cells. See Fig. 11B.
These purified Ig originated from the immunized mice also possess adoptive immunity. The process to study adoptive immunity is as follows: nude mice were injected subcutanously 1×10⁵-1×10⁶ tumor cells. One day later, purified Igs were intravenously injected in the dosage of 10-300mg/kg twice a week for three consecutive weeks. Results showed that the adoptive transfer of human EGFR recombinant DNA vaccine hEe-p has significant tumor suppression effect. As a comparison, purified Ig were incubated at 4□ with fixed EGFR-positive tumor cells or EGFR-native tumor cells for one hour of shaking and mixing in order for the tumor cells to adsorb the Ig. This process was repeated four times. Results showed that Ig from the mice immunized with human EGFR DNA vaccine hEe-p were pre-adsorbed by the EGFR-positive tumor cells, such as LL/2c and MA782/5S, and thus lost their anti-tumor effect, while the anti-tumor effect of the Ig still remained after incubating with EGFR-negative tumor cells, such as H22 (Fig. 11C).
Besides the lung cancer and mammary gland cancer as described above, the tumors that can be affected include other solid tumors wherein EGFR is over-expressed, including ovarian cancer, colorectal cancer, prostate cancer, stomach cancer, bladder cancer, head and neck squamocarcinoma and glioma. EGFR molecular vaccine has reliable anti-tumor effect on all cancers mentioned above.

7. Principle of the effect and biological safety of the EGFR molecular vaccine

It is well known in modern immunology that the mutual recognition between an antigen or antigenic epitope and its corresponding receptor/antibody is not merely specific. In fact, there is a certain degree of plasticity, promiscuity, and degeneracy in this recognition. Thus, it is possible to induce immune cross-reaction against a self-antigen through the mechanism of molecular mimicry in order to break the immuno-tolerance for the self-antigen.
As discussed above, the present invention discloses the discovery that tumor specific protein, such as EGFR molecule, is an immune cross-reactive antigen. Certain variation of the EGFR molecule can break the body's tolerance for self-EGFR molecule, and induce self cross-reactive immune response against the EGFR molecule. These immune response against EGFR include active immune response (such as cellular immune response and humoral immune response), and passive immune response (such as adoptive immune response).
In order to study the principle or mechanism of the anti-tumor function of EGFR molecular vaccine, we first undertook an immune testing for humoral liquid from EGFR immunized mice. The testing used included flow cytometry, Western Blot, immunoprecipitation in order to determine the existence of anti-EGFR autoantibody in mouse and rabbit serum. We used ELISA to test for the titer and type of anti-EGFR antibody in mouse/rabbit immunized sera, and we used immunohistochemistry to test the autoantibody in the tumor tissue of immunized mice. We used tumor cell aggregation test, and serum adoptive immune test for examining the funtion of autoantibody.
The following experimental protocols were undertaken. Blood was taken from each mouse before and after immunization every week using extraction of blood sample from the mouse tail vein, or after the mouse was sacraficed, sera were collected for use in the next step. Western Blot showed that antibodies induced by recombinant EGFR molecular vaccine could recognize specifically the corresponding antigen (recombinant EGFR protein or EGFR expression in tumor cells), but they could not recognize EGFR-negative cells. At the same time, flow cytometry was used to identify the antibody stereo surface recognition and we found that recombinant EGFR molecular vaccine could stimulate the production of specific antibodies which could in turn recognize the EGFR on the surface of tumor cells. Testing for autoantibodies using ELISA showed that the mice started to produce anti-mouse autoantibody two weeks after being immunized with recombinant EGFR molecular vaccine, reaching a titer of 1:100 to 1:5000. It gradualy increased to 1:10000 to 1:500000 at week 4, and could maintain a titer of 1:500 to 1:1000 at week 8. As a comparison, the corresponding control group had no detectable antibody production. Further testing for the subtype of the resultant antibody showed that antibody produced by EGFR molecular vaccine was mainly IgG. Fig. 12 shows the types of antibodies produced by the mice immunized with EGFR molecular vaccine (both recombinant DNA vaccine and recombinant Protein vaccine). From the figure, it can be seen that recombinant EGFR molecular vaccine can increase significantly the production of IgG1, IgG2a, and IgG2b, but that the production of IgM and IgA is not increased. Moreover, the production of the antibodies stimulated by recombinant EGFR molecular vaccine can be blocked by anti-CD4 antibody, but not by anti-CD8, anti-NK, or control antibodies. As comparison, no specific antibodies were detected in the control groups of protein vaccine, adjuvant, or saline.
EGFR molecular vaccine can also induce cellular immunity. The tests used to examine cellular immunity in the mice immunized with EGFR molecular vaccine include: using ⁵¹Cr release method to measure CTL activities; using ELISPOT to test cytokine level (mainly for testing serum concentration of IFN-γ, and IL-4); using depletion of immune cell subsets tests to determine T cell types (CD4⁺ T lymphocyte, CD8⁺ T lymphocyte, or NK cells, etc).
The protocols are carried out as follows: mice were immunized with recombinant chEGFR vaccine, and then spleen monolymphocyte were taken for ELISPOT test, which showed a large quantity of antigen specific B cells in the monolymphocyte. Testing in the mice immunized with recombinant mEGFR showed the existence of a small amount of antigen specific B cells. However, no statistically significant amount of antigen specific B cells was found in control groups including adjuvant group or blank vector group. In another experiment, spleen T cells were taken from the mice immunized with recombinant EGFR molecular vaccine for three weeks. Then, we used monolymphocyte from health spleen as antigen presenting cells to activate T cells in vitro in the presence of antigen. Results showed that the mice immunized with recombinant chEGFR molecular vaccine contained relatively a larger amount of IFN-γ, and IL-4 producing cells. Spleen T derived from recombinant chEGFR molecular vaccine immunized mice cells also produced many IFN-γ and IL-4 producing cells after being stimulated again in vitro by mEGFR molecular vaccine. Both groups of spleen T cells produced significantly more than T cells that were not stimulated in vitro. Furthermore, standard ⁵¹Cr tests were used to determine the specific cytocidal effect of spleen T cells immunized by recombinant EGFR molecular vaccine against the relevant tumor cells from human and mice. Results showed that after immunized by chEGFR molecular vaccine, at the ratio of 40:1, mouse spleen T cells exhibited specific cytocidal activity against EGFR positive tumor cells. In the example of LL/2c and MA 782/5S, they are 40.27%, 42.83%, respectively. In contrast, mice spleen T cells immunized with mEGFR exhibited no cytocidal effect on LL/2c and MA 782/5S tumor cells, and on EGFR-negative tumor cells, such as C26 cells. At the same time, the cytocidal effect mentioned above can be blocked by corresponding anti-CD8 and anti MHC-I monoclonal antibodies, but was not blocked by anti-CD4 and anti-MHC-II monoclonal antibodies. Spleen T cells from control groups did not show any statistically signifcant cytocidal activity. Figure 13 shows the CTL effect of EGFR recombinant DNA vaccine on immunized mice. It can be seen from this figure that T cells from mice immunized with human EGFR recombinant DNA vaccine hEe-p has higher cytocidal activity against EGFR-positive tumor cells compared to T cells from the control groups. Moreover, this in vitro cytocidal activity can be blocked by anti-CD8 or anti-MHC-I monoclonal antibody, but not by anti-CD4 monoclonal antibody, showing that this cytocidal activity is dependent on MHC-I dependent CD8⁺ T cells. In addition, the activated spleen cells exhibited no increase in NK activity against YAC-1 target cells. Moreover, adoptive transfer of CD4-depleted (CD8⁺), or CD8-depleted (CD4⁺) T cells derived from mice immunized with human EGFR recombinant DNA vaccine hEe-p exhibited anti-tumor effect against EGFR-positive tumor. In contrast, its anti-tumor effect is not significant against EGFR-negative tumor cells. No anti-tumor effect was seen from the control group.
We also observed a long-term potential toxic impact on the mice immunized with EGFR molecular vaccine. No obvious toxic side effects were observed, such as weight loss, skin and hair deteriaration, loss of apetitie, reduced life expectancy. Microscopic examination of the liver, lung, spleen, and brain of immunized mice uncovered no pathological changes. Immunofluorescent staining showed no deposition of autoantibody in major organs of the mice. See Fig. 14.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic diagram of the mechanism of action of homologous molecular vaccine.
Figure 2 shows recombinant EGFR plasmid maps. 2A shows recombinant EGFR eukaryotic expression plasmid maps; 2B shows recombinant EGFR prokaryotic expression plasmid maps; 2C shows recombinant EGFR yeast expression plasmid maps; 2D shows recombinant EGFR Adenovirus shuttle plasmid maps; 2E shows recombinant EGFR Lentivirus precursor plasmid maps.
Figure 3 is a flow chart depicting the construction of EGFR recombinant protein vaccine.
Figure 4 is a flow chart depicting the construction of EGFR recombinant virus vaccine. Figure 4A is a flow chart depicting the construction of EGFR recombinant Adenovirus vaccine; Figure 4B is a flow chart depicting the construction of EGFR recombinant Lentivirus vaccine.
Figure 5 is a flow chart depicting the construction of the RGD-modified EGFR recombinant Adenovirus vaccine.
Figure 6, including Figure 6A, 6B and 6C, is a schematic diagram depicting the mechanism of action for the nanoparticle targeted EGFR molecular vaccine.
Figure 7 is a graph showing the induction of protective anti-tumor immunity of EGFR recombinant DNA vaccine. 7A. Tumor Volume changes of immunized mice bearing LL/2c Lewis lung cancer. 7B. Tumor Volume changes of immunized mice bearing MA782/5S mammary cancer. 7C. Survival rate of immunized mice bearing LL/2c Lewis lung cancer. 7D. Survival rate of immunized mice bearing MA782/5S mammary cancer. hEe-p, human EGFR extracellular DNA vaccine; mEe-p, mouse EGFR extracelluar DNA vaccine; c-p, blank plasmid control; Saline, saline control.
Figure 8 is a graph showing the induction of therapeutic anti-tumor immunity of EGFR recombinant DNA vaccine. 8A. Tumor Volume changes of immunized mice bearing LL/2c Lewis lung cancer. 8B. Tumor Volume changes of immunized mice bearing MA782/5S mammary cancer. 8C. Survival rate of immunized mice bearing LL/2c Lewis lung cancer. 8D. Survival rate of immunized mice bearing MA782/5S mammary cancer, hEe-p, human EGFR extracellular DNA vaccine; mEe-p, mouse EGFR extracelluar DNA vaccine; c-p, blank plasmid control; Saline, saline control.
Figure 9 depicts the induction of anti-tumor immunity of EGFR recombinant protein vaccine. 9A. Protective immunity; 9B. Therapeutic immunity; 9C. Survival curves for mice bearing MA782/5S mammary cancer. edCER, Recombinant protein vaccine containing Chicken EGFR extracellular domain. edMER: Recombinant protein vaccine containing Mouse EGFR extracellular domain. Adj: Adjuvant. NS: Saline.
Figure 10 depicts the anti-tumor metastasis effect of EGFR recombinant protein vaccine. 10A. Number of transferer of LL/2c lung cancer. 10B. Lung wet weight after LL/2c tumor metastasis. edCER: Recombinant protein vaccine containing Chicken EGFR extracellular domain. edMER: Recombinant protein vaccine containing Mouse EGFR extracellular domain. Adj: Adjuvant. NS: Saline.
Figure 11 depicts EGFR molecular vaccine's inhibition of tumor cell growth in vitro, and its adoptive anti-tumor immunity. 11A. The inhibition of growth of EGFR-positive tumor cells (A549, LL/2c, MA782/5S) and EGFR-negative tumor cells (H22 and MMT-06052) by Ig derived from mice immunized with EGFR Recombinant DNA vaccine hEe-p. 11 B. Inhibition of growth of tumor cells by Ig derived from non-immunized normal mice. 11C. In vivo adoptive anti-tumor immunity of Ig derived from mice immunized with EGFR recombinant DNA vaccine hEe-p. hEe-p, human EGFR extracellular DNA vaccine; mEe-p, mouse EGFR extracelluar DNA vaccine; c-p, blank plasmid control; Saline, saline control.
Figure 12 depicts the types of antibodies induced by EGFR molecular vaccine. 12A. Types of antibodies induced by EGFR recombinant DNA vaccine.12B. Types of antibodies induced by EGFR recombinant protein vaccine. hEe-p, human EGFR extracellular DNA vaccine; mEe-p, mouse EGFR extracelluar DNA vaccine; c-p, blank plasmid control; Saline, saline control. edCER. Recombinant protein vaccine containing Chicken EGFR extracellular domain. edMER: Recombinant protein vaccine containing Mouse EGFR extracellular domain. Adj: Adjuvant. NS: Saline.
Fig.13 depecits the induction of CTL-mediated cytotoxicity in vitro with EGFR DNA vaccine and adoptive transfer of T cell subsets. A, T cells derived from the spleens of hEe-p-immunized mice were tested against LL/2c cells at different E:T ratios by a standard ⁵¹Cr release assay. T cells derived from the spleens of hEe-p-immunized mice showed higher cytotoxicity against LL/2c cells than did T cells from mEe-p, c-p, or nonimmunized mice by a standard ⁵¹Cr release assay. hEe-p-induced tumor killing activity can be blocked by anti-CD8 or anti- MHC class I (anti-H-2Kb/H-2Db) mAb. B and C, T cells were isolated from spleens of C57BL/6 mice, immunized with hEe-p (□), mEe-p, and nonimmunized mice (▪), and were depleted of CD4⁺ or CD8⁺ lymphocytes. The adoptive transfer of 2×10⁷ CD4-depleted (CD8⁺) (B) or CD8-depleted (CD4⁺) (C) T cells from mice immunized with hEe-p showed the antitumor activity against EGFrpositive LL/2c. D and E, The adoptive transfer of 2×10⁷ CD4-depleted (CD8⁺)(D) or CD8-depleted (CD4⁺) (E) T cells from BALB/c mice immunized with hEe-p was also found to be effective in EGFR-positive MA782/5S mammary cancer model. There was no antitumor activity found in syngeneic EGFr-negative tumor (B16 and Meth A) (B-E). In addition, the transfer of T lymphocyte subsets from mice immunized with mEe-p and nonimmunized mice (▪) had no effect (B-E). Data represent day 25 after tumor cell injection.
Fig.14 depicts the identification of auto-antibodies on the tumor cells by fluorescence microscopy. There was the deposition of auto-antibodies on LL/2c tumor cells (A) and MA782/5S tumor cells (B) from hEe-p-immunized mice, but not on the corresponding tumor cells from non-immunized mice (C and D). The mice depleted of CD4⁺T lymphocytes were immunized with hEe-p and did not develop detectable IgG-specific fluorescence on LL/2c tumor cells (E) and MA782/5S tumor cells the tumor cells (F). In contrast, the depletion of CD8⁺ lymphocytes (G and H) or NK cells showed no effect. There was no deposition of auto-antibodies within the tissues of the liver (I) and kidney (J) of mice immunized with hEe-p and the corresponding tissues (K and L) from non-immunized mice.

DETAILED EMBODIMENTS OF THE INVENTION

The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

Example One: EGFR Recombinant DNA Vaccine

GenBank was searched to obtain the cDNA sequences for human, mouse, Chicken, and they are listed herein as SEQ ID NO 1-5,7-9,19, respectively. We designed the following PCR primers based upon the cDNA sequences. For pORF based plasmids
We use the total RNA from human lung cancer cell line A431, mouse lung cancer cell line LL2, and Chick Embryo to undertake RT-PCR amplification, then collect and purify the amplified EGFR fragment (average 1.9kb in length) using electrophoresis, and then subclone the PCR products. After confirming the sequences of the PCR subclones through sequencing, we digest them using Ncol and EcoRI, collect the 1.9 kb fragments and purify them, and then insert the fragments into the pORF-MCS vector (from the InvivoGen Corporation) which had been digested with Ncol and EcoRI. We select recombiant plasmids. Candidate recombinant plasmids are confirmed by both restriction enzyme digestion analysis and PCR amplification. They are named pORF-hEGFR, pORF-mEGFR and pORF-chEGFR.

For pcDNA based plasmids:

For the construction of pcDNA-hEGFR, pcDNA-mEGFR
pcDNA-chEGFR, the process is similar to the above described process. In this case, we used pcDNA3.1 (+) vector made by InvitroGen, and vary the sequences of the PCR primers somewhat from those primers described in the above paragraph.
We selected recombinant expression plasmids, use restriction enzyme analysis to confirm the presence of the EGFR sequence, and then transfect them into CHO, NIH3T3, Vero cell lines, respectively. We monitor the level of EGFR expression using SDS-PAGE, ELISA, Western Blot techniques. We used alkaline extraction to harvest the recombinant EGFR expression plasmids. Then, we use ultracentrifuge and ultra-filtration to eliminate E. coli toxin contamination, and thus obtaining pure plasmid DNA. These plasmid DNAs can be used as DNA vaccine to immunize subjects, in this case, mice.
Similar to the above process, we can design other primers or probes according to the sequences collected in public databases such as GenBank, the sequences including gene, cDNA, mRNA and amino acid sequences. These sequences can be from a variety of species such as human, mouse, rat, chicken, mackeral, drosphia, etc, see SEQ ID Nos: 1-14. Using these sequences, we clone and isolate the extracellular domains of EGFR molecules from different biological sources, using PCR, RT-PCR, molecular hybridization techniques, using commercial gene libraries, cDNA libraries (such as ClonTech, StrateGene's gene libraries and cDNA libraries), and cell lines (such as human lung cancer cell line A431, mouse Lewis lung cancer line LL2, etc.), or tissues (such as lung cancer, breast cancer tissues, drosphia tissue, and chick embryo, etc.). Also, we obtained EGFR molecules possessing strong immunogenicity using selections with the gene-directed evolutionary technique. After confirming the EGFR cDNA extracellular sequences using sequencing, we inserted the EGFR extracellular domain into Eukaryotic expression plasmids such as pcDNA3.1, pORF-mcs, pBLAST-MCS, pSecTag2, etc.
We selected mice aged 6-8 weeks old, and established various mouse tumor models using routine techniques. We injected 100□g of EGFR recombinant expression plasmids, once a week, for 4 weeks continuously. We observed the growth of the tumor-bearing mice, and the progression of the tumor. After 8 weeks, the mice were sacrificed, and sera of the immunized mice and organs were collected. We used flowcytometry, ELISA, and Westem Blot to test for humoral immune response. To examine cellular response, we used Cr⁵¹, ELISpot methods. To determine toxicity and side effects of the experiments, we used immunohistochemistry. Next, we purified the sera of the immunized mice, used routine methods to establish tumor models in nude mice, and undertake adoptive immunotherapy, and further observe the growth of the tumors.

Example Two: EGFR Recombinant Protein Vaccine (Expressed in E. coli)

As in Example One, we obtain the cDNA sequences of EGFR from human, mouse, and chicken from public databases such GenBank, corresponding to SEQ ID NO 1-5,7-9,19, respectively. Based upon these sequences, we designed the following primers:
We use pORF-hEGFR, pORF-mEGFR pORF-chEGFR as template for PCR amplification, then collect and purify the amplified EGFR fragment (average 1.9kb in length) using electrophoresis, and then subclone the PCR products. After confirming the sequences of the PCR subclones through sequencing, we digest them using Ncol and BglII, collect the 1.9 kb fragments and purify them, and then insert the fragments into the pQE60 vector (from the Qiagen Corporation) which had been digested with two restriction enzymes Ncol and BgIII. We select recombiant plasmids. Candidate recombinant plasmids are confirmed by both restriction enzyme digestion analysis and PCR amplification. They are named pQE-hEGFR, pQE-mEGFR pQE-chEGFR.
Similarly, depending upon different prokaryotic expression vectors such as pET32, pLLp, pSE420, different primers can be designed and used to construct other EGFR recombinant prokaryotic expression plasmids.
Similar to Example One, we cloned the extracellular domain of various EGFR molecules, and then insert the domain into the prokaryotic expression plasmids such as pQE, pET32, pLLp, etc. After selecting recombinant expression plasmids, we confirmed the sequences of the domain using restriction enzyme digestion. We then transformed these recombinant plasmids into E.coli hosts such as E.coli TOP10F', E.coli BL21(DE3)pLys, E.coli M15, etc., and then observed and tested the level of expression of EGFR molecule using techniques such as SDS-PAGE, ELISA, Western Blot, etc. From these testing, we determined the optimal E.coli host for expression the recombinant plasmids. We then used confirmed EGFR expression plasmids to transform the optimal E.coli host cells, and then established stable transformants and various levels of seed bank libraries. We cultured the recombinant bacterial cells in shaking incubators, or alternatively, we fermented the bacteria using standard methods. We harvested the recombinant bacteria using low temperature centrifuge. Then we suspended the bacteria in PBS, and then broke the cells using ultrasound method. We used ionic exchange chromatography and affinity chromatography to isolate and purify the recombinant EGFR proteins. The purified EGFR proteins can be used as protein vaccine to immunize subject mice.
As in Example One, we selected mice aged 6-8 weeks old, and established various mouse tumor models using routine techniques. We injected 5-50□g of EGFR recombinant protein, once a week, for 4 weeks continuously. We observed the growth of the tumor-carrying mice, and the development of the tumor. After 8 weeks, the mice were sacrificed, and sera of the immunized mice and organs were collected. We used flowcytometry, ELISA, and Western Blot to test for humoral immune response. To examine cellular response, we used Cr⁵¹, ELISpot methods. To determine toxicity and side effects of the experiments, we used immunohistochemistry. Next, we purified the sera of the immunized mice, used routine methods to establish tumor models in nude mice, and undertake adoptive immunotherapy, and further observe the growth of the tumors.

Example Three: EGFR Recombinant Protein Vaccine (Expressed in Yeast Pichia pastoris)

Again, as described in the previous sections, we obtain the cDNA sequences of EGFR from human, mouse, and chicken from public databases such GenBank, corresponding to SEQ ID NO 1-5,7-9,19, respectively. Based upon these sequences, we designed the following primers.
We use pORF-hEGFR, pORF-mEGFR and pORF-chEGFR as template for PCR amplification, then collect and purify the amplified EGFR fragment (average 1.9kb in length) using electrophoresis, and then subclone the PCR products. After confirming the sequences of the PCR subclones through sequencing, we digest them using two restriction enzymes Xhol and Xbal (in the case of plasmids containing mouse EGFR, we use Xhol and NotI double enzyme digestion), collect the 1.9 kb fragments and purify them, and then insert the fragments into pPICZ A vector (from Invitrogen Corporation) which had been digested with two restriction enzymes Xhol and Xbal (in the case of plasmids containing mouse EGFR, we use Xhol and Notl double enzyme digestion). We transformed E.coli with these plasmid preparations, and select recombiant plasmids. Candidate recombinant plasmids are confirmed by both restriction enzyme digestion analysis and PCR amplification. They are named yeast expression plasmids: pYE-hEGFR, pYE-mEGFR and pYE-chEGFR.
After digesting these yeast expression plasmids with Pmel to linerize them, we use electric perforation method to transform yeast cell lines X33, or GS115. We use Zeocin resistance to select stable transformants. We use MMH (Minimal Methanol with histidine, MMH) and MDH (Minimal Dextrose with histidine, MDH) agar plate) to determine and selecte Mut⁺ transformants. We select 6-10 Mut⁺ transformants for small-scale expression, and then use SDS-PAGE, Western Blot, ELISA, etc. to confirm the expressed recombinant protein. We select the Mut⁺ transformants with the highest expression efficienty, and culture them at a large scale so as to establish yeast expression seed libraries. We use large flasks to culture or ferment the recombinant yeast cell lines, collect the yeast pellets using low temperature centrifuge. After resuspend the pellets n PBS solution, we use ultrasound to break the cells. Then, we used Ion Exchange Chromatography and affinity chromatography to isolate and purify the recombinant EGFR protein. The recombinant EGFR protein derived as such can be used as protein vaccine to immunize subjects. Similarly, yeast recombinant expression plasmids made with EGFR can be produced with other yeast expression systems.
As in Example Two, we selected mice aged 6-8 weeks old, and established various mouse tumor models using routine techniques. We injected 5-50□g EGFR recombinant protein, once a week, for 4 weeks continuously. We observed the growth of the tumor-carrying mice, and the development of the tumor. After 8 weeks, the mice were sacrificed, and sera of the immunized mice and organs were collected. We used flowcytometry, ELISA, and Western Blot to test for humoral immune response. To examine cellular response, we used Cr⁵¹, ELISpot methods. To determine toxicity and side effects of the experiments, we used immunohistochemistry. Next, we purified the sera of the immunized mice, used routine methods to establish tumor models in nude mice, and undertake adoptive immunotherapy, and further observe the growth of the tumors.

Example Four: EGFR Recombinant Adenovirus Vaccine

As in the previous examples, we obtained the cDNA sequences of EGFR from human, mouse, and chicken from public databases such GenBank, corresponding to SEQ ID NO 1-5,7-9,19, respectively. Based upon these sequences, we designed the following primers:
We use pORF-hEGFR, pORF-mEGFR and pORF-chEGFR as template for PCR amplification, then collect and purify the amplified EGFR fragment (average 1.9kb in length) using electrophoresis, and then subclone the PCR products. After confirming the sequences of the PCR subclones through sequencing, we digest them using two restriction enzymes BglII and EcoRV double enzyme digestion, collect the 1.9 kb fragments and purify them, and then insert the fragments into pShuttle-CMV vector (made by Quantum Biotechnologies) which was pre-digested with BglII and EcoRV. We select recombiant plasmids. The resulting candidate recombinant plasmids are confirmed by both restriction enzyme digestion analysis and PCR amplification. They are named Adenovirus shuttle expression plasmids: pShuttle-hEGFR, pShuttle-mEGFR and pShuttle-chEGFR. See Fig. 2D for plasmid maps of these Adenovirus shuttle expression plasmids.
We then take the various Adenovirus shuttle expression plasmids as described before, digest them with Pmel enzyme, and co-transform E.coli BJ5183 cells with backbone vector containing the Adenovirus genome pAdEasy-1 or pAdEast-2. The resulting recombinant Adenovirus vector plasmids are named pAd-hEGFR, pAd-mEGFR and pAd-chEGFR. These Adenovirus vector plasmids are digested with PacI enzyme, and then using the Calcium-phosphate-DNA coprecipitation method, they are transfected into the Adenovirus packaging cell line 293 cells. The resulting recombinant Adenoviruses are called Ad-hEGFR, Ad-mEGFR and Ad-chEGFR. PCR, Western blot and other methods are used to confirm that the EGFR gene has been indeed integrated into the Adenovirus vector, and that EGFR has been correctly, and efficiently expressed in Eukaryotic cells.
Depending upon the difference of the Adenovirus Genome, the recombinant Adenovirus vaccine can be classified into two groups: the first group is called Generation I of EGFR Recombinant Adenovirus, resulting from the recombination between Adenovirus Shuttle expression plasmid pShuttle-EGFR and AdEasy-1 recombinant, thus named Ad-hEGFR I, Ad-mEGFR I and Ad-chEGFR I. The second group is called Generation II of EGFR Recombinant Adenovirus, resulting from the recombination between Adenovirus Shuttle expression plasmid pShuttle-EGFR and AdEasy-2 recombinant, thus named Ad-hEGFR II, Ad-mEGFR II and Ad-chEGFR II.
We used ultracentrifuge to collect large quantity of the recombinant adenovirus Ad-EGFR, and then tested the titer (pfu) of each batch of the recombinant virus using upper layer agarose method, and TCID50 method. We then used 293 cells to produce large amount of confirmed EGFR adenovirus vaccine. We further isolated and purified the recombinant virus using ultracentrifuge and ultrafiltration. The purified EGFR recombinant adenovirus can be used a vaccine to immunize subject animals.
Similar to what's described in the previous examples, we selected mice aged 6-8 weeks old, and established various mouse tumor models using routine techniques. We injected 1×10⁹ PFU EGFR recombinant adenovirus in each mouse, once a week, for 4 weeks continuously. We observed the growth of the tumor-carrying mice, and the development of the tumor. After 8 weeks, the mice were sacrificed, and sera of the immunized mice and organs were collected. We used flowcytometry, ELISA, and Western Blot to test for humoral immune response. To examine cellular response, we used Cr⁵¹, ELISpot methods. To determine toxicity and side effects of the experiments, we used immunohistochemistry. Next, we purified the sera of the immunized mice, used routine methods to establish tumor models in nude mice, and undertake adoptive immunotherapy, and further observe the growth of the tumors.

Example Five: EGFR Recombinant Lentivirus Vaccine

As an illustration of the process of making EGFR Recombinant Lentivirus, we use the ViraPower™ Lentiviral Gateway® Expression Kit made by Invitrogen Corporation. The detailed process is as follows: we first search public database such as GenBank to obatin cDNA sequences of EGFR molecule of human, mouse and chicken. Their sequences are listed as SEQ ID NO 1-5,7-9,19, respectively.
We then designed the following PCR primers:
We use pORF-hEGFR, pORF-mEGFR and pORF-chEGFR as template for PCR amplification, then collect and purify the amplified EGFR fragment (average 1.9kb in length) using electrophoresis, and then subclone the PCR products. After confirming the sequences of the PCR subclones through sequencing, we digest them using two restriction enzymes Ncol and EcoRV double enzyme digestion, collect the 1.9 kb fragments and purify them, and then insert the fragments into pENTR11 vector (made by Invitrogen) which was pre-digested with Ncol and EcoRV. We select recombiant plasmids. The resulting candidate recombinant plasmids are confirmed by both restriction enzyme digestion analysis and PCR amplification. They are named as pENTR-hEGFR, pENTR-mEGFR and pENTR-chEGFR. See Fig. 2E for plasmid maps of these Lentivirus expression plasmids.
We then take the various Lentivirus expression plasmids as described before, co-transform E.coli DH5 cells together with backbone vector containing the Lentivirus genome pLenti6/V5-DEST. The resulting recombinant Lentivirus vector plasmids are named pLenti-hEGFR, pLenti-mEGFR and pLenti-chEGFR. See also Fig. 2E. We then mix these Recombinant Lentivirus vector plasmids with packaging mix, the ViraPower™ Packaging Mix, and then using the Calcium-phosphate-DNA coprecipitation method, the Recombinant Lentivirus vector plasmids are transfected into the Lentivirus packaging cell line 293FT cells. The resulting recombinant Lentivirus are called Lenti-hEGFR, Lenti-mEGFR and Lenti-chEGFR. PCR, Western blot and other methods are used to confirm that the EGFR gene has been indeed integrated into the Lentivirus vector, and that EGFR has been correctly, and efficiently expressed in Eukaryotic cells.
We used ultracentrifuge to collect large quantity of the recombinant adenovirus Ad-EGFR, and then tested the titer (pfu) of each batch of the recombinant virus using upper layer agarose method, and TCID50 method. We then used 293 cells to produce large amount of confirmed EGFR adenovirus vaccine. We further isolated and purified the recombinant virus using ultracentrifuge and ultrafiltration. The purified EGFR recombinant adenovirus can be used a vaccine to immunize subject animals.
Similar to what's described in the previous examples, we selected mice aged 6-8 weeks old, and established various mouse tumor models using routine techniques. We injected 1×10⁹ PFU EGFR recombinant Lentivirus in each mouse, once a week, for 4 weeks continuously. We observed the growth of the tumor-carrying mice, and the development of the tumor. After 8 weeks, the mice were sacrificed, and sera of the immunized mice and organs were collected. We used flowcytometry, ELISA, and Western Blot to test for humoral immune response. To examine cellular response, we used Cr⁵¹, ELISpot methods. To determine toxicity and side effects of the experiments, we used immunohistochemistry. Next, we purified the sera of the immunized mice, used routine methods to establish tumor models in nude mice, and undertake adoptive immunotherapy, and further observe the growth of the tumors.

Example Six: Mannan-modified Recombinant Adenovirus EGFR vaccine

The following procedure applies to the preparation of Mannan-modified Recombinant Adenovirus EGFR vaccine: we first use standard protocol to obtain and amplify EGFR Recombinant Adenovirus (either Generation I or II), and then use chromatography or ultracentrifuge to purify the recombinant adenovirus. Next, we dissolve 70mg Mannan from Sigma into 5ml 0.1 M phosphate buffer (pH6.0) to reach a final concentration of 14mg/ml, and then add 45ml 0.01M Sodium Periodate solution, and mix and oxidize at 4° C for 60 minutes. After that, we add 10µl glycol, and incubate for 30 minutes at 4° C, resulting in Oxidative Mannan (Ox-M) mixture.
We then load the Ox-M mixture onto Sephadex-G25 columns previously balanced with bicarbonate buffer (pH6.0-9.0) and perform chromatography, with Ox-M being eluted into 2ml sized empty vessel. After that, we mix the purified Ox-M with 1x10¹⁴ Recombinant Adenovirus particles at room temperature overnight, obtaining the needed Ox-M-Adenovirus. We then add 1mg/ml Sodium Borohydride to the Ox-M-Adenovirus, leave at room temperature for 3 hours, forming Reductive Mannan Adenovirus (Red-M-Adenovirus). Both Ox-M-Adenovirus and Red-M-Adenovirus are desalted by ultrafiltration, and condensed, filtering out bacteria. They are stored in small test tubes, and preserved at -80° C. The Mannan-modified Recombinant EGFR Adenovirus can be used as vaccine to immunize a subject.
Similar to what's described in the previous examples, we selected mice aged 6-8 weeks old, and established various mouse tumor models using routine techniques. We injected 1×10¹⁰ PFU EGFR recombinant Lentivirus in each mouse, once a week, for 4 weeks continuously. We observed the growth of the tumor-carrying mice, and the development of the tumor. After 8 weeks, the mice were sacrificed, and sera of the immunized mice and organs were collected. We used flowcytometry, ELISA, and Western Blot to test for humoral immune response. To examine cellular response, we used Cr⁵¹, ELISpot methods. To determine toxicity and side effects of the experiments, we used immunohistochemistry. Next, we purified the sera of the immunized mice, used routine methods to establish tumor models in nude mice, and undertake adoptive immunotherapy, and further observe the growth of the tumors.

Example Seven: RGD-modified Recombinant Adenovirus EGFR vaccine

In the present invention, we utilize the AdEasy system to construct the RDG modified Adenovirus Recombinant EGFR vaccine. The detailed process is shown step by step in Figure 5.
The detailed process is as follows: we digest the Adenovirus backbone plasmid pAdEasy-1 and pAdEasy-2 with restriction enzyme Spel (Sp), and then use T4 DNA Polymerase to fill in the ends (filling, f) so as to make them blunt, and then digest the filled-in product with PacI (P), and recover the 6211 bp and 3579bp fragments using electrophoresis, and name them AdFiber I/Sp/f/P and AdFiber II/Sp/f/P , respectively. These fragments contain the intact Adenovirus fiber protein gene. Separately, prepare the psuttle vector by digesting it with BamHI first, and then fill in with T4 DNA Polymerase, and then digest with PacI. After such BamHI/filling /PacI-digestion treatment, the vector is ready to be inserted with the AdFiber I/Sp/f/P AdFiber II/Sp/f/P fragments. The resulting plasmids are named pSh-AdFiber I and pSh-AdFiber II, respectively.
We then digest pSh-AdFiber I with Nhel enzyme, fill in with T4 DNA polymerase, and digest again with Kpnl enzyme ( NheI/filling/KpnI), recover, using electrophoresis, the 2090 bp fragment called AdFiber I/Nh/f/K; insert this fragment into a pUC18 vector which had been pre-digested with Smal and Kpnl double enzyme digestion, resulting in the recombinant plasmid named pUC-AdFiber I.
On the other hand, pSh-AdFiber II is digested with Avrll enzyme, then filled in with T4 DNA polymerase, and then digested with Hindlll (AvrII/filling/HindIII), using electrophoresis, recover the 838 bp fragment, called AdFiber I/A/f/H. Insert this fragment into a pUC vector which had been previously digested with SmaI and Hindlll double enzyme digestion, resulting in new plasmids named pUC-AdFiber II.
Next, we designed a series of PCR primers so as to use pUC-AdFiber I and pUC-AdFiber II as templates to amplify the Adenovirus knob, (Ad-knob) gene sequences. The primers used are, respectively:
Using primer F1-R1, F2-R2, F3-R1 and F2-R3, respectively, for the first round PCT, we obtain products PCR1, PCR2, PCR3 and PCR4. Again, using F1-R2 and F3-R3 as primers, and using the amplified products from the first round, PCR1 and PCR2, PCR3 and PCR4 as templates, we undertake the second round of PCR amplification, resulting in PCR products PCR1-PCR2 (PCR I) , PCR3-PCR4 (PCR II) . We take the PCR I and PCR II from the amplification in the second round, insert them into pBR322 vector that had been previously cut with EcoRV. The resulting recombinant plasmids are named pBR-PCR I and pBR-PCR II.
The sequence of the RGD-4C duplex is as follows:
We insert the RGD-4C into the pBR-PCR I and pBR-PCR II vectors, where the vector were previously digested with Smal. The resulting recombinant plasmids are named pBR-PCR/RGD I and pBR-PCR/RGD II. The sequences of the recombinant plasmids are confirmed using sequencing. Cut pBR-PCR/RGD I with NheI/KpnI double digestion, using electrophoresis, rcover the PCR/RGD I fragment. Insert the fragment into the pUC-AdFiber I vector which had been double digested using Nhel/Kpnl. The resulting recombinant plasmids are called pUC-AdFiber-RGD I.
Similarly, we use AvrII/HindIII to double digest pBR-PCR/RGD II, and using electrophoresis, recover the PCR/RGD II fragment. Again, insert the fragment into pUC-AdFiber II vectors previously digested with double enzume AvrII/HindIII, the resulting plasmid is named pUC-AdFiber-RGD II.
Afterwards, we use SpeI/PacI double enzyme to digest pUC-AdFiber-RGD I and pUC-AdFiber -RGD II vector, and using electrophoresis, recover the AdFiber-RGD I, and AdFiber -RGD II fragments. Insert the fragment into pAdEasy-1, pAdEasy-1 both of which were previously digested with SpeI/PacI, the resulting recombinant plasmids are called pAdEasy-RGD I, and pAdEasy-RGD II, respectively.
Next, we first take pShuttle-hEGFR, pShuttle-mEGFR and pShuttle-chEGFR as described above, and linerize them with Pmel. After that, we co-transform the E.coli BJ5183 cells with these Shuttle-EGFR plasmids together with pAdEasy-RGD I, and pAdEasy-RGD II, respectively. The resulting recombinant plasmids are named Adenovirus plasmids pAd-RGD-EGFR I, and pAd-RGD-EGFR II.
We used the Adenovirus plasmid pAd-RGD-EGFR I to transfect 394 cells, resulting in recombinant Adenovirus named Ad-RGD-EGFR I. Similarly, we transfect Adenovirus plasmid pAd-RGD-EGFR II to 293E4pIX cells, resulting in recombinant Adenovirus named Ad-RGD-EGFR II. After purification, Ad-RGD-EGFR I and Ad-RGD-EGFR II can be used as vaccine to immunize subjects, and more importantly, these vaccines have specificity targeting tumor vascular endothelial cells.

Example Eight: Pharmacological studies of Recombinant EGFR Molecular vaccine

It is important to obtain pharmacological studies relating to the anti-tumor effect of recombinant EGFR molecular vaccine. Studies can be performed for this purpose in the present invention include: ordinary tumor-bearing mice (including protective immunity study, therapeutic immunity study, and metastasis model study), rabbit immunological test, monkey immunological test, nude mice adoptive immunological test, dosage-dependency test, and in vitro cellular test, etc. In this example, we describe the ordinary tumor-bearing mice test, using lung cancer as a model. Other tests can be performed similarly.

1. Protective immunity of tumor-bearing mice:

6-8 weeks old C57BU6 mice are randomly divided into groups. They are injected EGFR molecular vaccine in both hind legs intramuscularly, once a week for four weeks continuously. At the 5^th week, mice were are challenged with LL/2c tumor cells in the concentration of 1x10⁶. Blood is taken from tail vein or from sacraficing the mice at week 0, 1, 3, 5 after initial immunization. After centrifuge (5000RPM for 3 minutes), sera is collected and stored at -20° C for future use.

2. Therapeutic immunity of tumor-bearing mice:

6-8 weeks old C57BL/6 mice are randomly divided into groups. They are challenged with LL/2 cancer cells at a concentration of 1x10⁶. Four days after tumor introduction, they are randomly divided into groups, and immunized with EGFR molecular vaccine by injection intramuscularly in the hind legs, once a week for four weeks continuously. At week 0, 2. 4, and 6 after initial immunization, blood is taken from tail vein or from sacraficing the mice. After centrifuge (5000RPM for 3 minutes), sera is collected and stored at -20° C for future use. Record tumor weight, volume and survial curves.

3. Metastasis model test of tumor-bearing mice:

6-8 weeks old C57BL/6 female mice are randomly divided into groups. They are injected EGFR molecular vaccine in both hind legs intramuscularly, once a week for four weeks continuously. After two weeks, LL/2 tumor cells at log growth period are selected, and injected intramuscularly into the hind legs of the mice, at a concentration of 2x10⁵ per mouse. Contiue the injection mentioned above. After four weeks, mice are killed, and their lungs are weighed. After examination for tumor transferer in the lung, the lungs are fixated with 10% neutrally buffered formaldehyde.
It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.

Claims

1. A method for making vaccine comprising the steps of:

1) analyzing specific antigen of particular pathogen;

2) obtaining a polynucleotide sequence encoding the specific antigen;

3) obtaining a polynucleotide sequence having a sufficient difference from the polynucleotide sequence; and

4) preparing the vaccine using the polynucleotide sequence obtained in step 3).

2. The method according to claim 1, wherein said pathogen is selected from the group of bacterial, virus, fungus and protozoan.

3. The method according to claim 1, wherein the homology between the polynucleotide sequence of step 1) and the polynucleotide sequence of step 3), as compared by the polypeptide sequences encoded by them, is 30 to 95%.

4. The method according to claim 1, wherein the form of said vaccine is selected from the group consisting of recombined protein vaccine, recombined gene vaccine, recombined virus vaccine, genetically modified vaccine, and stably inverted symbiotic bacteria.

5. A method for making cancer vaccine comprising the steps of:

1) analyzing specific antigen of tumor cell in an animal;

2) obtaining a polynucleotide sequence encoding the specific antigen;

4) preparing the vaccine using the polynucleotide sequence obtained in step 3).

6. The method according to claim 5, wherein said tumor cell occurs in an animal.

7. The method according to claim 5, wherein said tumor cell originates from an animal.

8. The method according to claim 6 or 7, wherein said animal includes human.

9. The method according to claim 5, wherein the homology between the polynucleotide sequence of step 1) and the polynucleotide sequence of step 3), as compared by the polypeptide sequences encoded by them, ranges from 30 to 95%.

10. The method according to claim 5, wherein the form of said vaccine is selected from the group consisting of recombined protein vaccine, recombined gene vaccine, recombined virus vaccine, genetically modified vaccine, and stably inverted symbiotic bacteria.

11. A method for making EGFR vaccine comprising the steps of:

1) analyzing EGFR antigen of tumor cell in an animal;

2) obtaining a polynucleotide sequence encoding the specific antigen;

4) preparing the vaccine using the polynucleotide sequence obtained in step 3).

12. The method according to claim 11, wherein said tumor cell occurs in an animal.

13. The method according to claim 11, wherein said tumor cell originates from an animal.

14. The method according to claim 11, wherein the homology between the polynucleotide sequence of step 1) and the polynucleotide sequence of step 3), as compared by the polypeptide sequences encoded by them, ranges from 30 to 95%.

15. The method according to claim 11, wherein the form of said vaccine is selected from the group consisting of recombined protein vaccine, recombined gene vaccine, recombined virus vaccine, genetically modified vaccine, and stably inverted symbiotic bacteria.

17. A nucleic acid vaccine, wherein the homology between the polypeptide encoded by the polynucleotide sequence contained in the vaccine and the polypeptide encoded by the polynucleotide sequence of specific antigen in pathogen is 30 to 95%.

18. An epidermal growth factor receptor (EGFR) nucleic acid vaccine, wherein the homology between the epidermal growth factor receptor encoded by the polynucleotide sequence contained in the vaccine and the amino acid sequence in the epidermal growth factor receptor of the organism is 30 to 95%.

19. An epidermal growth factor receptor (EGFR) nucleic acid vaccine, wherein the homology between the epidermal growth factor receptor encoded by the polynucleotide sequence contained in the vaccine and the amino acid sequence in the epidermal growth factor receptor of human is 30 to 95%.

20. A method for making EGFR vaccine used for treating human tumor comprising the steps of:

1) obtaining a polynucleotide sequence encoding EGFR from xenogeneic organism; and

2) preparing the vaccine by using the polynucleotide sequence obtained in step 1).

21. The method according to claim 20, wherein said xenogeneic organism is other organism except for human, such as mice, rat, chicken, mackerel, drosphia, and the like.

22. The method according to claim 20, wherein the polynucleotide sequence encoding EGFR from xenogeneic organism is selected from the group consisting of SEQ.ID.No.13, SEQ.ID.No.15, SEQ.ID.No.17, SEQ.ID.No.19, SEQ.ID.No.21, SEQ.ID.No.23, SEQ.ID.No.24, and SEQ.ID.No.26.

23. The method according to claim 20, wherein the form of said vaccine is selected from the group consisting of recombined protein vaccine, recombined gene vaccine, recombined virus vaccine, genetically modified vaccine, and stably inverted symbiotic bacteria.

24. The method according to claim 20, wherein the human tumor is selected from the group consisting of lung cancer, breast cancer, ovary cancer, colon cancer, prostate cancer, bladder cancer, head and neck squamocarcinoma and glioma, which over-express EGFR molecule.

25. A method for making EGFR vaccine used for treating human tumor comprising the steps of:

1) modifying the polynucleotide sequence of human EGFR; and

2) preparing the vaccine by selecting the modified polynucleotide sequence from the polynucleotide sequence obtained in step 1).

26. The method according to claim 25, wherein said polynucleotide sequence of human EGFR is selected from the group consisting of SEQ.ID.No.1, SEQ.ID.No.3, SEQ.ID.No.5, SEQ.ID.No.7, SEQ.ID.No.9, SEQ.ID.No.11.

27. The method according to claim 25, wherein said modification method is selected from the group consisting of error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis, gene reassembly, GSSM and any combination thereof.

28. The method according to claim 25, wherein the form of said vaccine is selected from the group consisting of recombined protein vaccine, recombined gene vaccine, recombined virus vaccine, genetically modified vaccine, and stably inverted symbiotic bacteria

29. The method according to claim 25, wherein the human tumor is selected from the group consisting of lung cancer, breast cancer, ovary cancer, colon cancer, prostate cancer, bladder cancer, head and neck squamocarcinoma and glioma, which over-express EGFR molecule.

30. An epidermal growth factor receptor polypeptide vaccine, wherein the homology between the epidermal growth factor receptor polypeptide contained in the vaccine and the amino acid sequence in the epidermal growth factor receptor of human is 30 to 95%.